Muscarinic agonists

ABSTRACT

The present invention relates to compounds of Formula I: Formula I which are agonists of the M-1 muscarinic receptor.

The present invention relates to the field of pharmaceutical and organicchemistry and provides compounds that are active at the muscarinicreceptors.

The compounds of the present invention are muscarinic agonists. Morespecifically, the compounds of the present invention are selectiveagonists of the muscarinic M-1 receptor. As such, they are useful fortreating a variety of disorders of the central nervous system and otherbody systems. These disorders include cognitive disorders, ADHD,obesity, Alzheimer's disease, psychoses including schizophrenia, and foralleviation of intraocular pressure such as that found in glaucoma.

Certain indane-like compounds are described as useful for treatingconditions associated with malfunctioning of the muscarinic cholinergicsystem in PCT Publication Nos. WO 97/25983, published 24 Jul. 1997, andWO 99/04778, published 4 Feb.1999.

The present invention provides compounds of Formula I:

wherein

-   -   Q, X, Y, and Z are independently selected from the group        consisting of CR¹ and N, provided that no more than two of Q, X,        Y, and Z are N and at least two of Q, X, Y, and Z are CH; or Y        is CH, Z is CH, and the moiety “Q=X” represents “S” to form a        thiophene ring;    -   R¹ is independently at each occurrence selected from the group        consisting of hydrogen, halogen, C₁-C₄ alkoxy, and C₁-C₄ alkyl;    -   R² is selected from the group consisting of halogen; C₁-C₄        alkoxy; C₁-C₄ alkyl; C₃-C₈ cycloalkyl; cyano; trifluoromethyl;        pyridinyl optionally substituted with one to two substituents        independently selected from the group consisting of halogen,        C₁-C₄ alkoxy, and C₁-C₄ alkyl; thienyl optionally substituted        with one substituent selected from the group consisting of        halogen, C₁-C₄ alkoxy, and C₁-C₄ alkyl; phenyl optionally        substituted with from one to three substituents independently        selected from the group consisting of halogen, C₁-C₄ alkoxy,        C₁-C₄ alkyl, trifluoromethyl, and cyano; and pyrrolyl optionally        substituted with one to two substituents independently selected        from the group consisting of halogen, C₁-C₄ alkoxy, and C₁-C₄        alkyl;    -   R³ is selected from the group consisting of phenyl optionally        substituted with one to three substituents independently        selected from the group consisting of halogen, C₁-C₄ alkoxy,        C₁-C₄ alkyl, trifluoromethyl, cyano, and nitro; naphthyl        optionally substituted with one to three substituents        independently selected from the group consisting of halogen,        C₁-C₄ alkoxy, C₁-C₄ alkyl, trifluoromethyl, cyano, and nitro;        heteroaryl optionally substituted with one or two substituents        independently selected from the group consisting of halogen,        C₁-C₄ alkoxy, and C₁-C₄ alkyl; or 1,3-benzodioxolyl optionally        substituted with one substituent selected from the group        consisting of halogen, C₁-C₄ alkoxy, and C₁-C₄ alkyl;    -   R⁴ is selected from the group consisting of hydrogen, hydroxy,        and fluoro;    -   R⁵ is selected from the group consisting of hydrogen, halogen,        C₁-C₄ alkoxy, and C₁-C₄ alkyl;    -   R^(a) is selected from the group consisting of hydrogen and        methyl;    -   t is one, two, or three; and    -   m is one or two;    -   or pharmaceutically acceptable addition salts thereof.

The present invention also provides pharmaceutical compositions,comprising a compound of Formula I and a pharmaceutically acceptablediluent.

Because the compounds of Formula I are agonists of the M-1 muscarinicreceptor, the compounds of Formula I are useful for the treatment of avariety of disorders associated with muscarinic receptors, including:cognitive disorders (including age-related cognitive disorder, mildcognitive impairment, cognitive impairment associated withschizophrenia, and chemotherapy-induced cognitive impairment), ADHD,mood disorders (including depression, mania, bipolar disorders),psychosis (in particular schizophrenia), dementia (including Alzheimer'sdisease, AIDS-induced dementia, vascular dementia, and dementia lackingdistinctive histology), Parkinson's disease, and Huntington's Chorea.Also, the present compounds are useful for treating chronic colitis,including Crohn's disease. Additionally, the present compounds areuseful for the treatment of pain (including acute pain and chronicpain), xerostomia (dry mouth), Lewy body disease (including diffuse Lewybody disease), aphasia (including primary aphasia and primary aphasiasyndromes), and hypotensive syndromes.

In another embodiment the present invention provides methods of treatingdisorders associated with muscarinic receptors, comprising:administering to a patient in need thereof an effective amount of acompound of Formula I. That is, the present invention provides for theuse of a compound of Formula I or a pharmaceutical composition thereoffor the manufacture of a medicament for the treatment of disordersassociated with muscarinic receptors. The present invention alsoprovides a compound of Formula I for use in therapy.

As used herein, the following terms have the meanings indicated:

The term “halogen” refers to a chloro, fluoro, bromo or iodo atom.

The term “C₁-C₄ alkyl” refers to a straight or branched alkyl chainhaving from one to four carbon atoms, and includes methyl, ethyl,n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, and t-butyl. Theterm “C₃-C₈ cycloalkyl” refers to cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cycloheptyl, and cyclooctyl.

The term “C₁-C₄ alkoxy” refers to a straight or branched alkyl chainhaving from one to four carbon atoms attached to an oxygen atom, andincludes methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, iso-butoxy,sec-butoxy, and t-butoxy.

The term “heteroaryl” is taken to mean a stable unsaturated five- orsix-membered ring containing from 1 to 2 heteroatoms selected from thegroup consisting of nitrogen, oxygen and sulfur. Examples of heteroarylinclude pyridinyl, pyrimidinyl, pyrazinyl, pyrrolyl, oxazolyl,isoxazolyl, imidazolyl, thiazolyl, pyridazinyl, furyl, thienyl, and thelike. Preferred heteroaryl groups are thienyl, pyridinyl, and furyl.

The compounds of the present invention form pharmaceutically acceptableacid addition salts with a wide variety of organic and inorganic acidsand include the physiologically acceptable salts which are often used inpharmaceutical chemistry. Such salts are also part of this invention. A“pharmaceutically-acceptable addition salt” is formed from apharmaceutically-acceptable acid as is well known in the art. Such saltsinclude the pharmaceutically acceptable salts listed in Journal ofPharmaceutical Science, 66, 2-19 (1977) which are known to the skilledartisan. Typical inorganic acids used to form such salts includehydrochloric, hydrobromic, hydriodic, nitric, sulfuric, phosphoric,hypophosphoric, metaphosphoric, pyrophosphoric, and the like. Saltsderived from organic acids, such as aliphatic mono and dicarboxylicacids, phenyl substituted alkanoic acids, hydroxyalkanoic andhydroxyalkandioic acids, aromatic acids, aliphatic and aromatic sulfonicacids, may also be used. Such pharmaceutically acceptable salts thusinclude chloride, bromide, iodide, nitrate, acetate, phenylacetate,trifluoroacetate, acrylate, ascorbate, benzoate, chlorobenzoate,dinitrobenzoate, hydroxybenzoate, methoxybenzoate, methylbenzoate,o-acetoxybenzoate, isobutyrate, phenylbutyrate, α-hydroxybutyrate,butyne-1,4-dicarboxylate, hexyne-1,4-di carboxylate, caprate, caprylate, cinnamate, citrate, form ate, fumarate, glycol late, heptanoate,hippurate, lactate, malate, maleate, hydroxymaleate, malonate,mandelate, mesylate, nicotinate, isonicotinate, oxalate, phthalate,teraphthalate, propiolate, propionate, phenylpropionate, salicylate,sebacate, succinate, suberate, benzenesulfonate,p-bromobenzenesulfonate, chlorobenzenesulfonate, ethyl sulfonate,2-hydroxyethylsulfonate, methylsulfonate, naphthalene-1-sulfonate,naphthalene-2-sulfonate, naphthalene-1,5-sulfonate, p-toluenesulfonate,xylenesulfonate, tartrate, and the like.

The present invention includes the stereoisomers and tautomers of thecompounds of Formula I. Herein, the Cahn-Prelog-Ingold designations of(R)- and (S)- and the cis and trans designation of relativestereochemistry are used to refer to specific isomers and relativestereochemistry.

As with any group of pharmaceutically active compounds, some groups arepreferred in their end use application. The following paragraphs definepreferred classes.

-   -   a) When R⁴ is not hydrogen, compounds which have trans        stereochemistry at the 1- and 2-position are preferred.    -   b) When R⁴ is not hydrogen, compounds which have the trans        stereochemistry at the 1- and 2-position shown below are more        preferred.

-   -   c) R^(a) is methyl.    -   d) R⁵ is hydrogen.    -   e) R⁴ is hydroxy.    -   f) t is one.    -   g) m is one.    -   h) R^(a) is methyl, R⁵ is hydrogen, R⁴ is hydroxy, t is one, and        m is one.    -   i) Q, X, Y, and Z are each CR¹ provided that at least two of Q,        X, Y, and Z are CH.    -   j) R¹ is hydrogen.    -   k) R¹ is halogen.    -   l) R¹ is fluoro.    -   m) Q, X, Y, and Z are each CH.    -   n) One of Q, X, Y, and Z is CF and the others are CH.    -   o) Q is CF and X, Y, and Z are each CH.    -   q) R² is phenyl optionally substituted with from one to three        substituents independently selected from the group consisting of        halogen, C₁-C₄ alkoxy, C₁-C₄ alkyl, trifluoromethyl, and cyano.    -   r) R² is phenyl.    -   s) R³ is phenyl optionally substituted with one to three        substituents independently selected from the group consisting of        halogen, C₁-C₄ alkoxy, C₁-C₄ alkyl, trifluoromethyl, cyano, and        nitro.    -   t) R³ is phenyl substituted with one substituent selected from        the group consisting of halogen, trifluoromethyl, cyano, or        nitro.    -   u) R³ is phenyl substituted once with halogen.    -   v) R³ is phenyl substituted once with fluoro.    -   w) R³ is phenyl substituted once with fluoro in the        para-position.    -   x) R² is phenyl, R³ is phenyl substituted once with fluoro in        the para-position, and Q, X, Y, and Z are each CH.    -   y) R² is phenyl, R³ is phenyl substituted once with fluoro in        the para-position, Q is CF, and X, Y, and Z are each CH.    -   z) R^(a) is methyl, R⁵ is hydrogen, R⁴ is hydroxy, t is one, m        is one, R² is phenyl, and Q, X, Y, and Z are each CH.    -   aa) R^(a) is methyl, R⁵ is hydrogen, R⁴ is hydroxy, t is one, m        is one, R² is phenyl, Q is CF, and X, Y, and Z are each CH.    -   bb) R^(a) is methyl, R⁵ is hydrogen, R⁴ is hydroxy, t is one, m        is one, and R³ is phenyl substituted once with fluoro in the        para-position.        The preceding paragraphs may be combined to define additional        preferred classes of compounds.

The compounds of Formula I in which R⁴ is hydroxy are prepared byprocedures described in Scheme A. In Scheme A all substituents, unlessotherwise indicated, are as previously defined, and all reagents arewell known and appreciated in the art.

In Scheme A, step a, the compound of Formula (1) is resolved to give asubstantially pure compound of Formula (2). The compound of Formula (1)is readily prepared by methods well known and appreciated in the art,such as those found in PCT Publication Nos. WO 97/25983, published 24Jul. 1997; and WO 99/04778, published 4 Feb. 1999. As used herein theterm “substantially pure” refers to enantiomeric purity. The desiredstereochemistry in final compounds of Formula 1 may be convenientlyintroduced in Scheme A, step a, by resolution of compounds of Formula(1). Further processing of resolved compounds of Formula (1), via stepsb, c, d, and optional step e, described infra, will result insubstantially pure compounds of Formula I. Substantially pure compoundsof Formula I can be prepared which are greater than 80%, preferablygreater than 90%, more preferably greater than 95%, most preferablygreater than 97% enantiomerically pure. The compound of Formula (1) canbe resolved by chiral chromatography or by fractional crystallization ofdiasteriomeric acid addition salts. It is expected that a wide varietyof such salts are suitable for this purpose. In practice, isomers ofmandelic acid have been found to be particularly useful.

For example, the compound of Formula (1) is contacted with the selectedacid. Generally, from about 0.4 molar equivalents to a large excess ofthe selected acid can be used with about 0.4 to 1.5 molar equivalentsbeing preferred and with about 0.5 to 1.1 molar equivalents being morepreferred. The resolution is typically carried out by crystallizing theacid addition salt from a solution. In particular, solvents such aslower alcohols, including methanol are useful. It may be advantageous touse small amounts of water with the selected solvent(s) in order tocarry out the resolution in a reasonable volume. The use of ananti-solvent may also be advantageous. As used herein, the term“anti-solvent” refers to a solvent in which the salt is significantlyless soluble compared to the other selected solvent(s). Preferably, whenan anti-solvent is used it is miscible with the other selectedsolvent(s). Suitable anti-solvents include ethers, such as diethylether, methyl t-butyl ether, and the like, and lower alkyl acetates,such as methyl acetate, ethyl acetate, isopropyl acetate, propylacetate, iso-butyl acetate, sec-butyl acetate, butyl acetate, amylacetate, iso-amyl acetate, and the like, and alkanes, such as pentane,hexane, heptane, cyclohexane, and the like. When the racemic mixture isused, care should be taken in using an anti-solvent to avoidcrystallization of the salt of the undesired diastereomeric salt.

Typically, the crystallization is carried out at initial temperatures ofabout 40° C. to reflux temperature of the selected solvent(s). Themixture is then cooled to give the salt. Seeding may be advantageous.Preferably the crystallization solution is cooled slowly. Thecrystallization is most conveniently cooled to temperatures of ambienttemperature to about −20° C. The salt can be collected using techniquesthat are well known in the art, including filtration, decanting,centrifuging, evaporation, drying, and the like. The compound of Formula(2) can be used directly as the acid addition salt of the selected acid.Alternately, before use the compound of Formula (2) can be isolated asanother acid addition salt after acid exchange or can by isolated as thebase by extraction under basic conditions as is well known andappreciated in the art.

As is readily apparent to one skilled in the art the depicted compoundof Formula (2) is of the trans configuration at the 1- and 2-positionsof the indane nucleus. Cis compounds are readily prepared from suchtrans compounds by protection of the amine, inversion of the hydroxycenter, followed by deprotection as needed. There are numerous methodswhich allow for inversions of hydroxy centers, such as by Mitsunobureaction with suitable carboxylic acids, including acetic acid andbenzoic acid, followed by hydrolysis.

Reaction Scheme A, step b, depicts the formation of a compound ofFormula (3). It is understood that the compound of Formula (3) can beone in which R is a group as desired in the final product of Formula Ias defined above. R may also combine with the carbonyl to form aprotecting group, such as t-BOC, which can be later removed beforeincorporation of an R group as desired in the final product of FormulaI. The selection and use of suitable protecting groups is well known andappreciated in the art (Protecting Groups in Organic Synthesis, TheodoraGreene (Wiley-Interscience)).

For example, where R is a group as desired in the final product, thecoupling reaction depicted in step b is carried out using theappropriate acid or the acid halide derived therefrom. Appropriate acidsinclude various substituted benzoic acids and acid halides, heteroarylacids and acid halides, and various biaryl carboxylic acids and acidhalides. Examples include biphenyl carboxylic acid and3-fluorobiphenyl-4-carboxylic acid.

For example, the compound of Formula (2) is contacted with anappropriate acid to give a compound of Formula (3). Such couplingreactions are common in peptide synthesis and synthetic methods usedtherein can be employed. For example, well known coupling reagents, suchas resin-bound reagents and carbodiimides with or without the use ofwell-known additives such as N-hydroxysuccinimide,1-hydroxybenzotriazole, etc. can be used to facilitate this acylation.The reaction is conventionally conducted in an inert aprotic polardiluent such as dimethylformamide (DMF), methylene chloride(dichloromethane), chloroform, acetonitrile, tetrahydrofuran (THF), andthe like. Typically the reaction is carried out at temperatures of fromabout 0° C. to about 60° C. and typically require from about 1 to about24 hours. Upon reaction completion, the product of Formula (3) isrecovered by conventional methods including extraction, precipitation,chromatography, filtration, trituration, crystallization and the like.

Alternatively, for example, the compound of Formula (2) is contactedwith an acid halide of an appropriate acid to give a compound of Formula(3). Such acid halides are commercially available or readily preparedfrom the corresponding acids by methods well known in the art, such asby the action of phosphorous trichloride, phosphorous tribromide,phosphorous oxychloride, phosphorous pentachloride, thionyl chloride,thionyl bromide, or oxalyl chloride, with or without a small amount ofdimethylformamide, in an inert solvent such as, toluene, methylenechloride, or chloroform; at temperatures of from about 0-80° C. Thereaction is typically carried out for a period of time ranging from 1hour to 24 hours. The acid halide can be isolated and purified or canoften be used directly, that is, with or without isolation and/orpurification. The coupling reactions generally use a suitable base toscavenge the acid generated during the reaction. Suitable bases include,by way of example, sodium hydroxide, potassium hydroxide, pyridine,triethylamine, N,N-diisopropylethylamine, N-methylmorpholine, and thelike. The reaction is conventionally conducted in a solvent such asmethylene chloride, chloroform, tetrahydrofuran and the like, or underSchotten-Baumann conditions in a solvent mixture such as methylenechloride, ethyl acetate, toluene and water. Typically the couplingreaction is carried out at temperatures of from about −20° C. to about80° C. and typically require from about 1 to about 24 hours. Uponreaction completion, the product of Formula (3) is recovered byconventional methods including extraction, precipitation,chromatography, filtration, trituration, crystallization and the like.

Reaction Scheme A, step c, depicts the reduction of a nitro group togive a compound of Formula (4). Such reductions can be carried out by avariety of methods that are well known in the art.

For example, a compound of Formula (3) may be hydrogenated over acatalyst, such as palladium-on-carbon, to give a compound of Formula(4). Such hydrogenations are generally carried out in a solvent and avariety of solvents are suitable, for example methanol, ethanol,isopropanol, tetrahydrofuran, or ethyl acetate or mixtures thereof. Thehydrogenation may be performed at an initial hydrogen pressure of 20-180psi (137-1241 kPa). The reaction is typically carried out at temperatureof about 0° C. to about 60° C. The reaction typically requires 1 hour to3 days. The product can be isolated and purified by techniques wellknown in the art, such as filtration, extraction, evaporation,trituration, precipitation, chromatography, and recrystallization.

In Scheme A, step d, a compound of Formula (4) is contacted with anappropriate amidine forming agent to give a compound of Formula I.Appropriate amidine forming agents include1-methylthio-1-methyl-N-(4-fluorobenzyl)-N-methylimmonium triflate and1-methylthio-1-methyl-N-(4-fluorobenzyl)-N-methylimmonium iodide. One ofordinary skill in the art will recognize that appropriate amidineforming agents may be prepared in advance or in situ if desired.

For example, a compound of Formula (4) is contacted with from about 1-3equivalents of an appropriate amidine forming agent. The reaction isgenerally carried out in a dry solvent such as methylene chloride,toluene, or tetrahydrofuran at temperatures of from about −20° C. to 50°C. The reaction is carried out using an appropriate base such aspyridine, collidine, or triethylamine. The reaction typically requires 1to 18 hours. The product can be isolated and purified by techniques wellknown in the art, such as quenching, filtration, extraction,evaporation, trituration, precipitation, chromatography, andrecrystallization.

As will be readily appreciated, where R is a protecting group introducedin step b, the protecting group can be removed after step d and theresulting amine coupled with an appropriate acid or acid halide as alsodescribed above in step b to give a compound of Formula I.

Some compounds of Formula I are intermediates for other final compoundsof Formula I. For example, when R² is iodo, another reagent, forexample, 2-(tributylstannyl)thiophene or 2-(tributylstannyl)pyridine,may be used to displace iodo as a leaving group and substitute adifferent R² group as desired in the final product.

In Scheme A, optional step e, not shown, an acid addition salt of acompound of Formula I is formed using a pharmaceutically-acceptableacid. The formation of acid addition salts is well known and appreciatedin the art.

The compounds of Formula I in which R⁴ is hydrogen are prepared fromcompounds of Formula (3) or from amine protected compounds of Formula(2) by deoxygenation. Such deoxygenation reactions are readily carriedout using procedures well known in the art, described, for example, byLarock, Comprehensive Organic Transformations, pg. 44-52 (1999).Alternately, the compounds of Formula I in which R⁴ is hydrogen areprepared by procedures described in Scheme B. In Scheme B allsubstituents, unless otherwise indicated, are as previously defined, andall reagents are well known and appreciated in the art.

Reaction Scheme B, step a, depicts the reductive amination of a compoundof Formula (5) to give a compound of Formula (6). Such reductiveaminations are carried out under a variety of conditions. The reactiondepicted in Scheme B, step a, can be carried out using ammonia or aprotected amine, such as benzyl amine, dibenzyl amine, and the likefollowed by deprotection to give the compound of Formula (6).

For example, a compound of Formula (5) is reacted with an excess ofammonia and sodium cyanoborohydride to give a compound of Formula (6).As is well known in the art, it may be advantageous to monitor andadjust the pH during such reactions. The reaction is carried out in asolvent, such as methanol, ethanol, isopropanol, and water or mixturesthereof. Typically the reaction is carried out at temperatures of fromabout 0° C. to about 60° C. and typically require from about 1 to about24 hours. Upon reaction completion, the product of Formula (6) isrecovered by conventional methods including extraction, precipitation,chromatography, filtration, trituration, crystallization, and the like.

Reaction Scheme B, steps b, c, d, and optional step e, are carried outby the methods described in Scheme A, steps b, c, d, and optional stepe, to give a compound of Formula I.

The compounds of Formula I in which R⁴ is fluoro are prepared fromcompounds of Formula (3) or from amine protected compounds of Formula(2) by halogenation procedures well known in the art, described, forexample, by Larock, Comprehensive Organic Transformations, pg. 689-701(1999).

The present invention is further illustrated by the following examplesand preparations. These examples and preparations are illustrative onlyand are not intended to limit the invention in any way.

The terms used in the examples and preparations have their normalmeanings unless otherwise designated. For example, “° C.” refers todegrees Celsius; “M” refers to molar or molarity; “mmol” refers tomillimole or millimoles; “g” refers to gram or grams; “mL” refersmilliliter or milliliters; “mp” refers to melting point; “brine” refersto a saturated aqueous sodium chloride solution; etc. In the ¹H NMR, allchemical shifts are given in δ, unless otherwise indicated.

Coupling Procedures

Method A

2′-Chlorobiphenyl-4-carboxylic acid

Combine methyl-4-bromobenzoate (1.0 g, 4.65 mmol), 2-chlorophenylboronicacid (799 mg, 5.1 mmol), Pd(OAc)₂ (51 mg, 0.46 mmol) and sodiumcarbonate (1.5 g, 13.9 mmol) in DMF (20 mL) and water (2.0 mL) withstirring. Purge the reaction mixture with argon, add triphenylphosphine(61 mg, 0.23 mmol) and purge again with argon. Place the sealed reactionin an oil bath maintained at 80° C. and allow to stir for 1 hour. Coolthe reaction to room temperature, dilute with ethyl acetate and filterthrough a short plug of celite with additional ethyl acetate. Wash theorganics with water, dry over MgSO₄, filter and evaporate. Purificationby flash column chromatography yields 2′-chlorobiphenyl-4-carboxylicacid methyl ester as a yellow solid. Dissolve the purified ester in THF(0.25M) and add an equal volume of 1M NaOH. Stir vigorously at roomtemperature for 15 hours. Upon completion, acidify the reaction withconc. HCl and extract with ethyl acetate. Evaporation of the solventyields 762 mg (67%) of the title compound. MS (m/e): 231.1 (M⁻).

The following compounds are prepared essentially as described above.

6-(2-Chlorophenyl)pyridine-3-carboxylic acid MS 233.9 (MH⁺)6-(2,4-Difluorophenyl)pyridine-3-carboxylic acid MS 235.9 (MH⁺)6-Phenylpyridine-3-carboxylic acid methyl ester MS 214.1 (MH⁺)6-(2-Methylphenyl)pyridine-3-carboxylic acid MS 214.0 (MH⁺)2′-Trifluoromethylbiphenyl-4-carboxylic acid MS 265.2 (M⁻)2-Methylbiphenyl-4-carboxylic acid MS 211.3 (M⁻)3-Fluorobiphenyl-4-carboxylic acid MS 215.1 (M⁻)2′,6′-Dichlorobiphenyl-4-carboxylic acid MS 264.9 (M⁻)2′,6′-Difluorobiphenyl-4-carboxylic acid MS 233.1 (M⁻)2′-Methoxybiphenyl-4-carboxylic acid MS 227.0 (M⁻)3,4′-Difluorobiphenyl-4-carboxylic acid MS 233.1 (M⁻)3,2′-Difluorobiphenyl-4-carboxylic acid MS 233.1 (M⁻)3-Chlorobiphenyl-4-carboxylic acid MS 231.1 (M⁻)4-(Thien-2-yl)phenyl-1-carboxylic acid MS 203.1 (M⁻)4′-Fluorobiphenyl-4-carboxylic acid MS 214.9 (M⁻) (Hydrolysis in dioxaneat 60° C.) 3′-Fluorobiphenyl-4-carboxylic acid MS 215.0 (M⁻) (Hydrolysisin dioxane) 3′-Cyanobiphenyl-4-carboxylic acid MS 222.0 (M⁻) (Hydrolysiswith LiOH in dioxane)

Method B

5-Phenylpyrazine-2-carboxylic acid

Combine 5-chloropyrazine-2-carboxylic acid methyl ester (626 mg, 3.64mmol), phenylboronic acid (666 mg, 5.45 mmol), cesium fluoride (55 mg,0.36 mmol) and Na₂CO₃ (964 mg, 9.09 mmol) in DMF (5 mL) and water (5 mL)with stirring. Place the hetereogeneous reaction mixture, open to theair, in an oil bath maintained at 80° C. After 5 minutes of heating, addPd(OAc)₂ (81 mg 0.36 mmol) in one portion and stir until reaction turnsblack. Cool the reaction to room temperature, dilute with ethyl acetate,and filter through a short plug of celite with additional ethyl acetate.Wash the organics with water, dry over MgSO₄, filter and evaporate.Purification by flash column chromatography yields2-phenylpyrimidine-5-carboxylic acid methyl ester as a yellow solid.Dissolve the purified ester in THF (0.25M) and add an equal volume of 1MNaOH. Stir vigorously at room temperature for 15 hours. Upon completion,acidify the reaction with conc. HCl and extract with ethyl acetate.Evaporation of the solvent yields 63 mg (8%) of the title compound. ¹HNMR (DMSO): 9.37 (s, 1H), 9.21 (s, 1H), 8.23-8.21 (m, 2H), 7.57-7.77 (m,3H).

The following compounds are prepared essentially as described above.

2′-Fluoro-6′-trifluoromethylbiphenyl-4-carboxylic acid MS 283.1 (M⁻)3,2′,4′-Trifluorobiphenyl-4-carboxylic acid MS 251.1 (M⁻)4′-Fluoro-2′-methoxybiphenyl-4-carboxylic acid MS 245.1 (MH⁻)3-Chloro-2′,4′-difluorobiphenyl-4-carboxylic acid MS 267.1 (M⁻)4′-Fluoro-2′-methylbiphenyl-4-carboxylic acid MS 229.0 (M⁻)4′-Trifluoromethylbiphenyl-4-carboxylic acid MS 265.1 (M⁻)2-Fluoro-4-(thien-2-yl)phenyl-1-carboxylic acid MS 221.1 (M⁻)

Method C

3′,4′-Difluorobiphenyl-4-carboxylic acid

Combine 3,4-difluorobenzeneboronic acid (1.0 g, 5.2 mmol),methyl-4-bromobenzoate (0.241 g, 1.73 mmol), Pd(OAc)₂ (0.019 g, 0.086mmol), tetrabutylammonium bromide (0.111 g, 0.345 mmol), and potassiumphosphate (0.733 g, 3.454 mmol). Purge the reaction vessel with argonand add anhydrous DMF (20 ml) to the reaction mixture. Heat the sealedreaction vessel to 120° C. with stirring until completion. Cool thereaction to room temperature, dilute with ethyl acetate, and filterthrough a short plug of celite with additional ethyl acetate. Washorganics with water, dry over MgSO₄, filter, and evaporate. Purificationby flash column chromatography yields3′,4′-difluorobiphenyl-4-carboxylic acid methyl ester as a yellow solid.Dissolve the purified ester in dioxane (45 ml) and add an equal volumeof 1 M aqueous NaOH. Heat the reaction vessel to 60° C. with stirringuntil completion. Remove the solvent by evaporation. Dissolve theresidue in dichloromethane and wash with 1N aqueous hydrochloric acid.Dry the organics over MgSO₄, filter and evaporate to yield 0.048 g (12%)of the title compound. MS (m/e): 235 (M⁺).

The following compounds are prepared essentially as described above.

6-(2-Fluorophenyl)pyridine-3-carboxylic acid MS 218.0 (MH⁺)3′,5′-Dimethylbiphenyl-4-carboxylic acid MS 225.0 (M⁻)3′,5′-Difluorobiphenyl-4-carboxylic acid MS 233.0 (M⁻)3′,5′-Dichlorobiphenyl-4-carboxylic acid MS 267.1 (M⁺)3′-Chlorobiphenyl-4-carboxylic acid MS 230.9 (M⁻)2′,3′-Difluorobiphenyl-4-carboxylic acid MS 264.9 (M⁻)4′-Chlorobiphenyl-4-carboxylic acid MS 230.9 (M⁻)

Method D

2′,4′,6′-Trimethylbiphenyl-4-carboxylic acid

Combine 1-iodo-2,4,6-trimetbylbenzene (2.966 g, 12.05 mmol),4-carboxyphenylboronic acid (1.0 g, 6.026 mmol), Pd(OAc)₂ (0.0067 g,0.005 mmol), tetrabutylammonium bromide (0.388 g, 1.2055 mmol), andpotassium phosphate (2.557 g, 12.05 mmol). Purge the reaction vesselwith argon and add anhydrous DMF (20 ml) to the reaction mixture. Beatthe sealed reaction vessel to 120° C. with stirring until completion asdetermined by TLC. Cool reaction mixture to room temperature. Add methyliodide (1.0 ml, 36.63 mmol) to reaction mixture with continued stirringuntil completion. Dilute the reaction with ethyl acetate and filterthough a short plug of celite with additional ethyl acetate. Wash theorganics with water, dry over MgSO₄, filter and evaporate. Purificationby flash column chromatography yields2′,4′,6′-trimethylbiphenyl-4-carboxylic acid methyl ester as a yellowsolid. Dissolve the purified ester in dioxane (45 ml) and water (5 ml)containing 5 eq of LiOH with stirring at 60° C. Upon completion,evaporate the solvent, acidify the reaction mixture with hydrochloricacid, and extract with ethyl acetate. Dry the organics over MgSO₄,filter, and evaporate to yield 0.023 g (16%) of the title compound. MS(m/e): 239.1 (M⁻).

The following compounds are prepared essentially as described above.

2′,4′,6′-Trifluorobiphenyl-4-carboxylic acid MS 251.0 (M⁻)2′-Fluoro-4′-Trifluoromethylbiphenyl-4-carboxylic acid MS 283.0 (M⁻)

Method E

2′,4′-Difluorobinhenyl-4-carboxylic acid

Combine 4-carbomethoxyphenylboronic acid (1.021 g, 5.67 mmol),1-bromo-2,4-difluorobenzene (1.000 g, 5.181 mmol.), Pd(OAc)₂ (0.113 g,0.50 mmol), triphenylphosphine (0.149 g, 0.505 mmol), and sodiumcarbonate (1.664 g, 0.568 mmol). Purge the reaction vessel with argon.Add DMF (20 mL) and water (2.0 mL) with stirring. Place sealed reactionin an 80° C. oil bath and allow to stir for 24 hours. Cool reaction toroom temperature, dilute with ethyl acetate, and filter through a shortplug of celite with additional ethyl acetate. Wash organics with water,dry over MgSO₄, filter, and evaporate. Purification by flash columnchromatography yields 2′,4′-difluorobiphenyl-4-carboxylic acid methylester as a yellow solid. Dissolve the purified ester in dioxane (5 ml)and add 5M NaOH (1 ml). Stir vigorously at 50° C. for 15 hours. Uponcompletion, acidify the reaction with conc. HCl and extract with ethylacetate. Evaporation of the solvent yields 300 mg (24.7%) of the titlecompound. MS (m/e): 233.0 (M⁻).

Method F

6-(2,6-Difluorophenyl)pyridine-3-carboxylic acid

Dissolve 6-chloropyridine-3-carboxylic acid methyl ester (6.86 g, 40mmol) in toluene (100 mL) and heat to 90° C. Add phosphorous oxybromide(25 g, 87 mmol) in several portions and continue heating for 3 hours.Cool the reaction to room temperature and pour onto ice water. Extractthe reaction with ethyl acetate and wash organics again with water thenNaHCO₃. Combine organics, dry over MgSO₄, filter, and evaporate toorange solid (8.1 g, 94%) which is an 8:1 mixture of6-bromopyridine-3-carboxylic acid methylester:6-chloromopyridine-3-carboxylic acid methyl ester by ¹H NMR.

Combine the mixture as obtained above (0.225 g, 1.04 mmol) withhexamethylditin (0.375 g, 1.15 mmol), Pd(OAc)₂ (21 mg, 0.09 mmol), andtriphenylphosphine (25 mg, 0.09 mmol) in toluene (5 mL). Purge with N₂and stir at 80° C. for 18 hours. Cool reaction to room temperature. Adda solution of 1-bromo-2,6-difluorobenzene (250 mg, 1.29 mmol) in toluene(1 mL) followed by Pd(OAc)₂ (21 mg, 0.09 mmol) and triphenylphosphine(25 mg, 0.09 mmol). Purge with N₂ and stir at 80° C. for an additional18 hours. Cool reaction to room temperature. Evaporate the solvent andpurify by column chromatography (silica, 10% ethyl acetate in hexane) togive 50 mg (20% yield) of 6-(2,6-difluorophenyl)pyridine-3-carboxylicacid ethyl ester. Hydrolyze the ester with 1 N sodium hydroxide solution(0.22 mL, 0.22 mmol) in methanol (3 mL) at room temperature for 3 days.Remove the volatiles under vacuum and combine the residue with 1 Nhydrochloric acid solution. Collect the white solid by filtration, washwith water, and dry under vacuum to give 30 mg (63% yield) of the titlecompound. MS (m/e): 235.9 (MH⁺).

Method G

3-Fluorobiphenyl-4-carboxylic acid

Combine methyl 2-fluoro-4-bromobenzoate (1.25 g, 5.36 mmol),phenylboronic acid (1.30 g, 10.72 mmol) and CsF (2.02 g, 13.40 mmol) inDMF (25 mL) and water (3.0 mL) with stirring. Place the hetereogeneousreaction mixture open to the air in an oil bath maintained at 80° C.After 5 minutes of heating, add Pd(OAc)₂ (120 mg, 0.536 mmol) in oneportion and stir until reaction turns black. Cool reaction to roomtemperature, dilute with ethyl acetate and filter through a short plugof celite with additional ethyl acetate. Wash organics with water, dryover MgSO₄, filter and evaporate. Purification by flash columnchromatography yields 3-fluorobiphenyl-4-carboxylic acid methyl ester asa solid. Dissolve the purified ester in THF (0.25M) and add an equalvolume of 1 M NaOH. Stir vigorously at room temperature for 15 hours.Upon completion, acidify the reaction with conc. HCl and extract withethyl acetate. Evaporation of the solvent yields 965 mg (84%) of thetitle compound. MS (m/e): 214.9 (M⁻).

The following compounds are prepared essentially as described above.

3-Fluoro-2′-methylbiphenyl-4-carboxylic acid MS 229.0 (M⁻)2′-Chloro-3-fluorobiphenyl-4-carboxylic acid MS 205.1 (M⁻)3-Fluoro-2′-trifluoromethylbiphenyl-4-carboxylic acid MS 283.1 (M⁻)

Method H

2-Fluoro-6-phenylpyridine-3-carboxylic acid

Dissolve 2,6-difluoropyridine (5.0 mL, 5.51 mmol) in anhydrous THF (30mL) and cool to −40° C. Add a solution of phenyl lithium (1.8 M hexanes,30.6 mL) dropwise over 5 minutes. Stir the resulting purple reaction at−40° C. for 30 minutes and bring to room temperature. Quench thereaction with water and extract the solution with ethyl acetate severaltimes. Combine the organic extracts, dry over MgSO₄, filter andevaporate onto silica gel. Purification by flash column chromatographyyields 2-fluoro-6-phenylpyridine 1.0 g (12%) as a yellow oil.

Cool a solution of LDA (3.46 mmol) in anhydrous THF (6 mL) to −78° C.Cannulate the 2-fluoro-6-phenylpyridine in anhydrous THF (6 mL) to thecooled LDA solution. Stir at −78 (C for 30 minutes then bubble carbondioxide gas through the solution for 10 minutes. Allow the reaction tocome to room temperature and purge with argon. Extract the reaction with1 M NaOH and discard the organics. Acidify the aqueous layer with conc.HCl and extract with ethyl acetate. Dry the organic layer over MgSO₄,filter and evaporate to yield the title compound as a light yellow solid(405 mg, 65%). MS (m/e): 216.1 (M⁻).

Method J

3,5-Difluorobiphenyl-4-carboxylic acid

Combine 1-bromo-3,5-difluorobenzene (0.863 mL, 7.50 mmol) andphenylboronic acid (1.22 g, 10.00 mmol) and subject to conditionsdescribed in Method G to yield 1.3 g of 3,5-difluorobiphenyl.

Dissolve crude 3,5-difluorbiphenyl (1.3 g, 6.83 mmol) in THF (14 mL) andcool to. −78° C. Prepare LiTMP from the addition of BuLi (1.6 M soln inhexanes, 5.33 mL) to tetramethyl piperidine (1.4 mL, 1.25 equiv) at −78°C. in THF (14 mL). Cannulate the cooled LiTMP into the cooled3,5-difluorobiphenyl and stir the reaction at −78° C. for 1 h. Bubblecarbon dioxide gas through the solution for 5 minutes, warm the reactionto rt, pour into 50 mL of 1M NaOB, and extract with 50 mL EtOAc. Discardthe organic layer was discarded. Acidify the remaining aqueous layerwith conc. HCl and extract twice with EtOAc. Dry the organics overMgSO₄, filtered, and evaporate to give 1.22 g of the title compound as awhite solid (77%). MS (m/e): 233.1 (M⁻).

Method K

3,2′,6′-Trifluorobiphenyl-4-carboxylic acid

Combine methyl 4-bromo-2-fluorobenzoate (3.66 g, 15.75 mmol),4,4,5,5,4′,4′,5′,5′-octamethyl-2,2′-bi-1,3,2-dioxaborolanyl (5.0 g,19.68 mmol) and potassium acetate (4.63 g, 47.19 mmol) in DMSO (40 mL)and purge the solution with argon. AddPdCl₂(1,1′-bis(diphenylphosphino)ferrocene)₂ (10 mol %, 1.35 g) andpurge the solution with argon again. Heat the reaction to 80° C. for 3 hand cool to room temperature. Wash the reaction with water and extractwith ethyl acetate and concentrate. The resulting black oil isre-dissolved in 1:2 ethyl acetate:hexanes, filtered through a short plugof silica gel, and concentrated.2-Fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoic acidmethyl ester is obtained as a yellow oil.

Dissolve the resulting yellow oil in acetone (100 mL) and combine withNalO₄ (10.1 g, 47.25 mmol), NH₄OAc (3.63 g, 47.25 mmol), and water (50mL) at room temperature. Stir at room temperature for 18 h, transfer toa separatory funnel and extract with ethyl acetate several times. Drythe combined organics over MgSO₄, filter and concentrate to yield 3.0 gof 3-fluoro-4-carbomethoxybenzene boronic acid as an off-white solid.

The boronic acid obtained above (800 mg, 4.04 mmol) and2,6-difluorobromobenzene (1.17 g, 6.06 mmol) are coupled according tothe procedure described in Method G to give 380 mg of the titlecompound. MS (m/e): 251.1 (M⁻¹).

Method L

6-Phenylpyridazine-3-carboxylic acid

6-Phenylpyridazin-3-ol (5.0 g, 29.06 mmol) is dissolved in toluene (100mL) and heated to 900° C. Phosphorous oxybromide (25 g, 87.19 mmol) isadded in several portions and the reaction is heated for 30 minutes. Theresulting yellow solution is cooled to room temperature, poured onto icewater, and extracted with ethyl acetate. The organic layers are furtherwashed with water and 1M NaOH, dried over MgSO₄, filtered, andevaporated to a yellow solid. Recrystallization from CHCl₃ gives 2.17 gof 3-bromo-6-phenylpyridazine.

3-Bromo-6-phenylpyridazine (1.0 g, 4.25 mmol) is combined with DMF (5mL), MeOH (5 mL), triethylamine (1.18 mL, 8.50 mmol), and Pd(OAc)₂ (76mg, 0.33 mmol) and the mixture evacuated.1,1′-Bis(diphenylphosphino)ferrocene (235 mg, 0.42 mmol) is added andthe reaction is again evacuated. Carbon dioxide gas is bubbled throughthe solution for 5 minutes, and the reaction is placed under 50 psi (345kPa) of carbon dioxide. The resulting solution is heated at 50° C. for18 h. The reaction is cooled to room temperature, diluted with water,and extracted with ethyl acetate. The organics are dried over MgSO₄,filtered, and evaporated onto silica gel and subjected to flash columnchromatography.

Hydrolysis using conditions outlined in Method A gives 80 mg of thetitle compound. ¹H NMR (CDCl₃): 8.24 (d, 1H, J=8.8 Hz), 8.18-8.15 (m,2H), 8.0 (d, 1H, J=9.2 Hz), 7.56-7.55 (m, 3H).

Method M

6-(4-Fluorophenyl)pyridine-3-carboxylic acid

Combine 6-bromopyridine-3-carboxylic acid methyl ester (1.03 g, 4.78mmol), 4-fluorophenylboronic acid (1.88 g, 13.41 mmol), and cesiumfluoride (2.55 g, 16.78 mmol) in DMF (25 mL) and water (4 mL) withstirring. Place the hetereogeneous reaction mixture, open to the air, inan oil bath maintained at 80° C. After 5 minutes of heating, addPd(OAc)₂ (150 mg, 0.67 mmol) in one portion. After 17 hours, cool thereaction to room temperature, dilute with ethyl acetate and filterthrough a short plug of celite with additional ethyl acetate. Wash theorganics with water, dry over MgSO₄, filter and evaporate. Purificationby flash column chromatography yields6-(4-fluorophenyl)pyridine-3-carboxylic acid methyl ester as a yellowsolid. Dissolve the purified ester in THF (0.25M) and add an equalvolume of 1M NaOH. Stir vigorously at room temperature for 15 hours.Upon completion, acidify the reaction with conc. HCL and collect thewhite precipitate by filtration. Drying under vacuum yields 385 mg (37%)of the title compound. MS (m/e): 218.1 (MH⁺)

The following compound is prepared essentially as described above.

6-(Thien-2-yl)pyridine-3-carboxylic acid MS 205.9 (MH⁺)

Method N

6-(4-Fluoro-2-methylphenyl)pyridine-3-carboxylic acid

Combine 6-bromopyridine-3-carboxylic acid methyl ester (387 mg, 1.79mmol), 4-fluoro-2-methylphenylboronic acid (338 mg, 2.19 mmol), Pd(OAc)₂(40 mg, 0.18 mmol), cesium fluoride (27 mg, 0.18 mmol) and sodiumcarbonate (570 mg, 5.38 mmol) in DMF (6 mL) and water (6 mL) withstirring. Purge the reaction mixture with N₂, add triphenylphosphine (47mg, 0.18 mmol), and purge again with N₂. Place the sealed reaction in anoil bath maintained at 80° C. and allow to stir for 17 hours. Cool thereaction to room temperature and pass through a short plug of silicagel. Wash the column with dichloromethane (100 mL) followed by aqueousmethanol (100 mL, 3 methanol/1 water). Reduce the combined fractions invacuo and suspend the residual solid in water (10 mL). Filter to removea black solid and acidify with 1 N hydrochloric acid solution to pH 4. Awhite precipitate forms which is collected by filtration and dried togive 306 mg (74%) of the title compound. MS (m/e): 231.9 (MH⁺).

The following compounds are prepared essentially as described above.

6-(2,4-Difluorophenyl)pyridine-3-carboxylic acid MS 236.0 (MH⁺)6-(2-Fluorophenyl)pyridine-3-carboxylic acid MS 218.0 (MH⁺)2′-Fluorobiphenyl-4-carboxylic acid MS 215.1 (M⁻)2′-Methylbiphenyl-4-carboxylic acid MS 211.2 (M⁻)

EXAMPLE 1-1 Biphenyl-4-carboxylic acid(R)-(6-(1-((4-fluorobenzyl)methylamino)ethylideneamino)-2(R)-hydroxyindan-1-yl)amide

Slowly add a solution of 375 g (5.13 mol, 1.12 equiv) ofN-methylacetamide in THF (1.76 L) to 224 g (5.55 mol, 1.2 equiv) ofsodium hydride (60% dispersion in mineral oil) as a slurry in THF (8.75L). After 30 minutes when 25% of the solution has been added, add 875 g(4.63 mole, 1 equiv) of 4-fluorobenzylbromide and the remainingN-methylacetamide and 4-fluorobenzylbromide solutions concurrently overthe next 3 h. Use a water bath to maintain the temperature below 40° C.Stir the resulting mixture overnight and pour into a mixture of 20%NH₄Cl (2.5 L), water (6.5 L), and ethyl acetate (9 L). Separate thelayers and back-extract the aqueous layer with ethyl acetate (4.5 L,then 2 L). Combine the organic layers and wash with water (4 L) and thenbrine (7 L). Dry the organic layer (Na₂SO₄) and remove the solvent toafford a residue. Dissolve the residue in acetonitrile (7 L) and heptane(1.75 L). Separate the layers and wash the acetonitrile layer again withheptane (1.75 L). Combine the heptane layers and back-extract withacetonitrile (0.5 L). Combine the acetonitrile layers and evaporate toafford 0.814 kg of N-methyl-N-(4-fluorobenzyl)acetamide.

Dissolve N-methyl-N-(4-fluorobenzyl)acetamide (0.500 kg, 2.76 mol) inTHF (11.5 L). Add phosphorus pentasulfide (0.737 kg, 1.65 mol, 0.6equiv) and heat the mixture to reflux over 1-2 hours. After 5 h atreflux, allow the mixture to cool to room temperature, filter off thesolids, and wash with 12.5 L of THF. Combine the filtrate with anidentical filtrate from a separate reaction and concentrate to 0.978 kgof a residue. Dissolve the residue and chromatograph on 2.7 kg of silicagel using CH₂Cl₂ to afford 1.01 kg of solid. Slurry the solid withmethylene chloride (1 L) for 15-30 min, add heptane (5 L), cool themixture to 0-5° C., and stir for 2 h. Collect the solid by filtrationand dry to afford 0.814 kg of N-methyl-N-(4-fluorobenzyl)thioacetamide.

Add 11.5 L of acetonitrile and 2.52 kg (17.7 mol, 1.5 equiv) of methyliodide to 2.30 kg (11.6 mol) of N-methyl-N-(4-fluorobenzyl)acetamide.Heat the mixture to 35° C. for 21 h. Reduce the volume by half on arotary evaporator and add 14 L of MTBE. Reduce the volume again by halfand add another 14 L of MTBE. Cool the resulting slurry to 0° C.,collect the solid by filtration, and dry to afford 3.92 kg of1-methylthio-1-methyl-N-(4-fluorobenzyl)-N-methylimmonium iodide as awhite solid.

Add 85 L of concentrated NH₄OH and 28 L of water to 6.20 kg (35.0 mol)of 1,2-epoxy-6-nitroindane. Heat the mixture at 36° C. for 21 h andallow to cool to room temperature. Filter the reaction mixture over abed of wet Celite (10 kg) and rinse the cake with water. Add to the wetcake 155 L of methanol, 1.3 L of water, and 5.80 kg (38.1 mol, 1.09equiv) of (S)-(+)-mandelic acid. Heat the mixture for 2 h at 55° C. andfilter through a carbon impregnated filter cartridge. Reduce thefiltrate volume by vacuum distillation to about 35 L and add 130 L ofEtOAc. Reduce the volume by vacuum distillation to about 65 L. Cool themixture to −8° C. and stir for 8 h. Filter the slurry and dry the solidto afford 7.6 kg of a solid. Slurry this solid in 30 L of methanol and0.3 L of water, and heat the mixture at reflux for 0.5 h. Cool themixture to 45° C. over 0.5 h and stir for 12 h, followed by cooling to22° C. and stirring for 10 h. Collect the solid by filtration and dry toafford 2.7 kg of 1(R)-amino-2(R)-hydroxy-6-nitroindane (S)-mandelate.

Add 1(R)-amino-2(R)-hydroxy-6-nitroindane (S)-mandelate (0.64 kg, 1.85mol) to a mixture of toluene (9.6 L) and aqueous 1 N NaOH (4.8 L, 4.8mol, 2.6 equiv). After 1 h, add 4-biphenylcarbonyl chloride (0.44 kg,2.0 mol, 1.1 equiv) in portions over 20-30 min. After 22 hours, filterthe solids under vacuum and rinse sequentially with 0.5 L of toluene, 2L of water, and 2 L of toluene. Dry the cake to afford 0.74 kg ofbiphenyl-4-carboxylic acid (R)-(6-nitro-2-hydroxyindan-1-yl)amide. Add38.2 L of ethyl acetate to 1.914 kg of biphenyl-4-carboxylic acid(R)-(6-nitro-2(R)-hydroxyindan-1-yl)amide prepared in a similar manner.Stir the slurry for 18 h, collect the solid by filtration, dry to afford1.76 kg of biphenyl-4-carboxylic acid(R)-(6-nitro-2(R)-hydroxyindan-1-yl)amide as a white solid.

Combine a slurry of 0.176 kg of 10% Pd-C (50% water wet) and 1.7 kg ofbiphenyl-4-carboxylic acid (R)-(6-nitro-2(R)-hydroxyindan-1-yl)amide in17.5 L of DMF with hydrogen (50 psi, 345 kPa). After 19 h, filter thereaction mixture, add a portion of the DMF solution (5 L) to water (10L), and stir the slurry for 2 h-repeat twice to work up the entirereaction volume. Filter the slurries together, and wash the resultingfilter cake with water (3×7 L). Dry the filter cake to afford 1.42 kg ofbiphenyl-4-carboxylic acid (R)-(6-amino-2(R)-hydroxyindan-1-yl)amide.

Slurry biphenyl-4-carboxylic acid(R)-(6-amino-2(R)-hydroxyindan-1-yl)amide (0.969 kg, 2.81 mol) in THF(9.7 L) and add1-methylthio-1-methyl-N-(4-fluorobenzyl)-N-methylimmonium iodide (0.954kg, 2.81 mol) and 4-dimethylaminopyridine (34.5 g, 0.281). Stir themixture for 24 h, and remove the solvent in vacuo. Dissolve theresulting foam in CH₂Cl₂ (12.5 L) and wash the organic phase with 1.0 NHCl (1×4 L and 1×3 L), 1.0 M NaOH (1×2.4 L) and saturated NaCl (1×4 L).Separate the organic phase, dry (Na₂SO₄), filter, and remove the solventto yield a solid. Dissolve the solid in acetonitrile (9 L) while heatingto 35-40° C. After approximately 30 minutes, add seed crystals, whichresults in a thick, white slurry. Cool the mixture to −15° C. and stirat this temperature for 1-2 h. Filter the slurry and dry to provide 1.10kg of the title compound as a partial acetonitrile solvate.

¹H NMR (CDCl₃): δ 7.90 (d, 2, J=8.6), 7.69 (d, 2, J=8.6), 7.63 (d, 2,J=8.2), 7.48 (t, 2, J=8.2, 7.6), 7.41 (d, 1, J=7.3), 7.24 (dd, 2, J=8.5,5.2), 7.14 (d, 1, J=7.9), 7.04 (t, 2, J=8.7), 6.72-6.63 (m, 3), 5.31 (t,1, J=5.6), 4.84 (br s, 1), 4.64 (dd, 2, J=21.4, 15.6), 4.54 (dd, 1,J=14.0, 7.9), 3.32 (dd, 1, J=15.6, 7.9), 3.01 (s, 3), 2.95 (dd, 1, J=15.7, 8.0), 1.97 (s, 3). MS (m/z): 508.2 (M+1).

EXAMPLE 2-1 Biphenyl-4-carboxylic acid(R)-(6-(1-((4-fluorobenzyl)methylamino)ethylideneamino)-2(R)-hydroxyindan-1-yl)amide

Combine trans-1-amino-2-hydroxy-6-nitroindane (20.2 g, 0.10 mol) andS-mandelic acid (16.7 g, 0.11 mol, 1.1 equiv) in 173 mL of methanol and3.6 mL of water. Heat at reflux and then add 200 mL of ethyl acetate.Seed and allow to cool to 23° C. After stirring for 4 h at 23° C., coolfor 3 hr at −3° C., then filter and rinse with cold 40% methanol and 60%ethyl acetate to give a solid. Dry the solid in a vacuum oven at 45° C.for 16 h to afford a 59:41 mixture of diastereomeric salts favoring1(R)-amino-2(R)-hydroxy-6-nitroindane.

Combine the 59:41 mixture of diastereomeric salts with 35 mL of methanoland 0.32 g of water. Heat to about 64° C., allow to cool to about 45°C., and stir for 14 h and then at 23° C. for 14 h to give a solid.Collect the solid by filtration, rinse with methanol, and dry in avacuum oven at 45° C. for 26 h to afford 2.64 g of 1(R)-amino-2(R)-hydroxy-6-nitroindane S-mandelate of high enantiomericpurity. HRMS (m/z): 194.0691. IR (CHCl₃) 1347, 1074 cm⁻¹.

Combine 1(R)-amino-2(R)-hydroxy-6-nitroindane S-mandelate 40.2 g (116.1mmol), 320 mL of water, 650 mL of ethyl acetate, 26.6 g ofdi-tert-butyldicarbonate (121.9 mmol, 1.05 equiv), and an additional 200mL of ethyl acetate. Add 120 mL of 1 N aqueous sodium hydroxide (120mmol, 1.03 equiv) dropwise. After 15 h, a solid forms.

Collect the solid and rinsed with water (3 times) and ethyl acetate (3times). Dry to give1(R)-(t-butoxycarbonylamino)-2(R)-hydroxy-6-nitroindane as white solid19.4 g (57%): MS (m/z): 295 (M+1). [α]D=−111 (c=1, MeOH).

Combine 1 (R)-(t-buloxycarbonylamino)-2(R)-hydroxy-6-nitroindane 30.5 g(0.11 mol), 800 mL of THF, 800 mL of ethyl acetate and 6.3 g of 5%palladium on carbon. Hydrogenate at 50 psi (345 kPa) of hydrogen for 2h. Remove the catalyst by filtration and evaporate the solvent to give29.5 g 1(R)-(t-butoxycarbonylamino)-2(R)-hydroxy-6-aminoindane: MS(m/z): 265 (M+1). IR (KBr) 1699, 1625, 1535 cm⁻¹. [α]D=−122 (c=1, MeOH).

To a solution of NaH (6.86 g, 0.172 mole, 60% in mineral oil, 1.3 equiv)in THF (250 mL), add dropwise a solution of N-methylacetamide (11.6 g,0.159 mole, 1.2 equiv) in THF (90 mL). After about 20 min, add4-fluorobenzylbromide (25 g, 0.132 mole). Stir for about 62 hours thenpour over ice water (300 mL) and extract with ethyl acetate (400 mL and200 mL). Combine the organic layers and wash with water (300 mL), dryover Na₂SO₄, filter and concentrate to an oil. Dissolve the oil inacetonitrile and extract with hexane to remove the mineral oil to give22.79 g N-methyl-N-(4-fluorobenzyl)acetamide: mp=48-54° C.; R_(f)=0.45(4% MeOH/methylene chloride); ¹H NMR (CDCl₃) 7.27-6.93 (m, 4), 4.5 (d,2), 2.91 (s, 3), 2.14 (s, 3).

Combine N-methyl-N-(4-fluorobenzyl)acetamide (364.8 g, 2.01 mol) and THF(9 L). Stir to afford a solution, then add phosphorus pentasulfide(537.7 g, 1.21 mol). After 45 min, heat to reflux. After 3 h, allow tocool and stir overnight to give a solid. Remove the solid by filtrationand rinse the filter cake with THF (4 L). Evaporate the filtrate to givea residue, dissolve in methylene chloride (500 mL) and passed over ashort column of silica gel 60 (1.2 kg) preconditioned with heptane.Elute with 4 L of heptane/methylene chloride (1:1) followed by 100%methylene chloride to afford, after collection and drying, 217.98 g ofN-methyl-N-(4-fluorobenzyl)thioacetamide: mp=99-104° C.; Rf=0.42(methylene chloride); ¹H NMR (CDCl₃) 7.35-7.26 (m, 1), 7.14-6.96 (m, 3),5.28 (s, 1.2) and 4.79 (s, 0.8), 3.42 (s, 1.2) and 3.15 (s, 1.8), 2.72(s, 1.2) and 2.69 (s, 1.8). Note: Partial protons are believed to be dueto rotomers.

Combine N-methyl-N-(4-fluorobenzyl)thioacetamide (14.03 g, 0.0711 mole)and methylene chloride (140 mL) under nitrogen and cool in an ice waterbath. Add dropwise methyl trifloromethanesulfonate (9.66 mL, 0.085 mole,1.2 equiv). After 15 min, remove the ice bath and stir for 2 hr. Removethe solvent under vacuum to obtain 25.89 g (100%) of1-methylthio-1-methyl-N-(4-fluorobenzyl)-N-methylimmonium triflate.

Combine 1-methylthio-1-methyl-N-(4-fluorobenzyl)-N-methylimmoniumtriflate (5 g, 13.8 mmol), methylene chloride (50 mL) and1(R)-(t-butoxycarbonylamino)-2(R)-hydroxy-6-aminoindane (3.65 g, 13.8mmol) under nitrogen. Add pyridine (0.15 mL). After 2.5 hr a solid wasformed. Collect the solid was by filtration, rinse with a minimal amountof methylene chloride, and dry in a vacuum oven to give 6.29 g (79%) of1(R)-(t-butoxycarbonylamino)-2(R)-hydroxy-6-(1-methyl-N′-(4-fluorobenzyl)-N′-methylamidino)indanetriflate as a white solid. mp=123-132° C.

Cool trifluoroacetic acid (TFA, 66 mL) in an ice bath and add1(R)-(t-butoxycarbonylamino)-2(R)-hydroxy-6-(1-methyl-N′-(4-fluorobenzyl)-N′-methylamidino)indanetriflate (29.65 g, 0.051 mole) in portions along with 3 mL of methylenechloride. Stir in the ice bath for 10 min and then allow to warm roomtemperature and stir for 1.5 hr. Partition the reaction mixture betweenmethylene chloride (500 mL) and ice water (500 mL). Add 2M aqueoussodium hydroxide (450 mL) and separate the layers. Extract the aqueouslayer with methylene chloride (250 mL) and the combined organic layerswere washed with water (300 mL). Cool the organic layer, add 1N aqueoussodium hydroxide (76 mL) and water (76 mL) and stir to give.

To theN′-(3-amino-2-hydroxyindan-5-yl)-N-(4-fluorobenzyl)-N-methylacetamidineproduced by the above procedures, add 4-biphenylcarbonyl chloride (11.05g, 0.051 mole, 1 equiv) in portions. Add an additional 50 mL of water.After 2 h, separate the layers, extract the organic layer with water(300 mL), dry over Na₂SO₄, filter and evaporate to give 27.06 g of thetitle compound as a white foam.

Crystallize a 10 g portion of the title compound from 10 mL/g ofacetonitrile to afford 5.96 g of the title compound: mp 103-111° C. MS(m/z): 508 (M+1).

Slurry the title compound obtained by recrystallization above in amixture of 45 mL of ethyl acetate and 45 mL of hexane for 16 h to affordthe title compound mp 139-142° C.

Combine the title compound having mp 139-142° C. (1 g) and absoluteethanol (12 mL). Heat to about 50° C. until the solids dissolve. Adddeionized water (4.5 mL) dropwise followed by the addition of seedcrystals of the polymorph melting at about 150-152° C. Cool to about 23°C. over 1.5 h, to give a thick suspension. Cool the suspension in an icebath, filter, and dry in a vacuum oven at 50-60° C. to yield 0.76 g ofthe title compound: mp 150-152° C.

As will be understood by one of ordinary skill in the art, an alternatename for the title compound is6-(1-((4-fluorobenzyl)methylamino)ethylideneamino)-2-hydroxy-1-biphenylamidoindane.

EXAMPLE 2-2 Biphenyl-4-carboxylic acid(R)-(6-(1-((4-fluorobenzyl)methylamino)ethylideneamino)-2(R)-hydroxyindan-1-yl)-amide

Combine 1(R)-amino-2(R)-hydroxy-6-nitroindane S-mandelate 50.0 g (0.144mole) and 750 mL of toluene. Add 375 mL (0.375 mole, 2.6 equiv) of 1Naqueous sodium hydroxide followed by 470 mL of water. Stir for about 22minutes then add 35.5 g (0.159 mole, 1.1 equiv) of 4-biphenylcarbonylchloride portion-wise over about 6 minutes. After 3.5 h, filter theresulting slurry, rinse the filter cake with toluene, dry in vacuo at50° C. for 18 h to give 56.1 g of1(R)-(4-biphenylcarbonylamino)-2(R)-hydroxy-6-nitroindane: 1R (KBr,cm⁻¹): 3293, 1640, 1549, 1528, 1345, 1329, 1086, 739; HRMS calc'd forC₂₂H₁₈N₂O₄:374,1267, Found: 374,1266.

Combine 1 (R)-(4-biphenylcarbonylamino)-2(R)-hydroxy-6-nitroindane 55.7g, 550 mL of DMF and 2.8 g of 10% Pd/C catalyst. Hydrogenate at 50 psi(345 kPa) of hydrogen at ambient temperature for 4.75 h. Filter thereaction mixture and dilute the filtrate with 1200 mL of water to give aslurry. Stir the slurry for 30 minutes, filter, wash with water, and dryin vacuo at 50° C. Combine the dried product and about 1 L of water andslurry for 30 minutes, filter, wash with water, and dry in vacuo at 50°C. to afford 45.4 g (90%) of 1(R)-(4-biphenylcarbonylamino)-2(R)-hydroxy-6-aminoindane: 1R (KBr,cm⁻¹); 3584, 3364, 3277, 1632, 1543, 1326, 1073, 743; HRMS calc'd forC₂₂H₂₀N₂O₂: 344,4120; Found: 344,1525.

Combine N-methyl-N-(4-fluorobenzyl)acetamide (364.8 g, 2.01 mol) and THF(9 L). Stir to dissolve and then add phosphorus pentasulfide (537.7 g,1.21 mol). After 45 min heat to reflux for 3 h, then cool and stirovernight to give a solid. Collect the solid by filtration and rinse thecake with THF (4 L), evaporate the filtrate in vacuo to give a residue,dissolve the residue in methylene chloride (about 500 mL), and passedover a cake of silica gel 60 (1.2 kg) preconditioned with heptane. Elutewith 4 L of heptane/methylene chloride (1:1) followed by 100% methylenechloride to afford 217.98 g of N-methyl-N-(4-fluorobenzyl)thioacetamide(55%): mp=99-104° C.; Rf=0.42 (methylene chloride); ¹H NMR (CDCl₃)7.35-7.26 (m, 1), 7.14-6.96 (m, 3),5.28 (s, 1.2) and 4.79 (s, 0.8), 3.42(s, 1.2) and 3.15 (s, 1.8), 2.72 (s, 1.2) and 2.69 (s, 1.8). Note:Partial protons are believed to be due to rotomers.

Add methyl iodide (10.76 g, 75.8 mmol) in one portion to a suspension ofN-methyl-N-(4-fluorobenzyl)thioacetamide (10 g, 50.6 mmol) inacetonitrile (50 mL). Heat at 35° C. for 46 h, then cooled to about 23°C. Reduce the volume of the reaction mixture to about 25 mL byevaporation and then add methyl t-butyl ether (50 mL). Again reduce thevolume by evaporation to 25 mL again and then dilute with another 50 mLof methyl t-butyl ether to give a solid. Cool to 0° C., filter, rinsewith 15 mL of cold methyl t-butyl ether, and dry to yield 16.89 g of1-methylthio-1-methyl-N-(4-fluorobenzyl)-N-methylimmonium iodide as ayellow solid, mp 142-150° C. MS (Electrospray): theoretical for iminiumportion C₁₁H₁₅FNS: 212; Found: 212.

Combine 1(R)-(4-biphenylcarbonylamino)-2(R)-hydroxy-6-aminoindane 10.0 g(0.029 mol), 4-dimethylaminopyridine (DMAP) 0.4 g (0.0032 mol, 0.011equiv), 200 mL of acetone and1-methylthio-1-methyl-N-(4-fluorobenzyl)-N-methylimmonium iodide 10.8 g(96% potency, 0.0305 mol, 1.05 equiv). After 6 h, concentrate thereaction mixture to a foam. Combine the foam and 120 mL of toluene, stirat ambient temperature for 19.5 h, filter, dry in vacuo at 50° C. togive a residue, the hydroiodide salt of the title compound,characterized by the following NMR: ¹H NMR (CDCl₃,300 MHz): δ 8.02 (d,J=9.0 Hz, 2H); 7.82-7.93 (m,1H); 6.90-7.69 (m, 14H); 4.85-5.25 (m, 2H);4.60-4.80 (m, 2H); 3.45 (s, 2H); 3.00-3.20 (m, 2H); 2.60-2.75 (m, 1H);2.10-2.35 (m, 4H).

Dissolve the residue in 200 mL of methylene chloride and extract with200 mL of 1.0 M aqueous sodium hydroxide followed by 200 mL of brine.Dry the organic layer over MgSO₄, filter, and evaporate in vacuo toafford 15 g of the title compound.

Combine 5.0 g of the compound obtained above and 200 mL of methylenechloride. Add 1.00 g of DARCO carbon, stir for 1 h, and then filterthrough a bed of Hyflo. Evaporate in vacuo to afford 4.0 g of a residue.Dissolve the residue in absolute ethanol (about 40 mL) and add deionizedwater (12 mL) to give a solid. Heat the slurried solid to 55° C. for 30min, allow to cool to room temperature, and stirred. After 24 h, filter,rinse with 10 mL of a 10:3 EtOH:water mixture, dry in vacuo to afford1.5 g of the title compound: mp 108-112° C.

One of ordinary skill in the art will recognize that an alternate namefor the title compound is6-(1-((4-fluorobenzyl)methylamino)ethylideneamino)-2-hydroxy-1-biphenylamidoindane.

EXAMPLE 3-1 3-Fluorobiphenyl-4-carboxylic acid(R)-(6-(1-((4-fluorobenzyl)methylamino)ethylideneamino)-2(R)-hydroxyindan-1-yl)amide

Combine 4-bromo-2-fluorobenzoic acid (10 g, 45.7 mmol), methanol (100mL), and concentrated sulfuric acid (5.0 mL). Heat to reflux. After 16h, cool to room temperature and evaporate in vacuo to afford a whitesolid. Dissolve the solid in ethyl acetate (about 40 mL), extract with2×80 mL saturated aqueous sodium bicarbonate and 1×80 mL brine, dry overMgSO₄, filter, and evaporate in vacuo to yield 9.32 g (88%) of methyl4-bromo-2-fluorobenzoate as a white solid. ¹H NMR (d₆-DMSO, 300 MHz)8.80 (t, J=8.4 Hz, 1H), 7.72 (dd, J=10.8 Hz, 1.8 Hz, 1H), 7.54 (ddd,J=8.4 Hz, 1.8 Hz, 0.6 Hz, 1H), 3.83 (s, 3H); IR (cm⁻¹, KBr): 3010, 2995,1723, 1603, 1484, 1438, 1406, 1294, 1277, 1095, 885; Anal calc'd forC₈H₆BrFO₂: C, 41.23; H, 2.60; Found: C, 40.97; H, 2.61.

Combine methyl 4-bromo-2-fluorobenzoate (9.3 g, 40.0 mmol),phenylboronic acid (9.75 g, 80.0 mmol), and cesium fluoride (32.6 g,100.1 mmol), DMF (190 mL) and deionized water (50 mL). Heat to 80° C.and add Pd(OAc)₂ (303 g, 4.0 mmol). After 20 min, cool to roomtemperature, filter through HyFlo with the aid of 100 mL ethyl acetate,extract the filtrate with 2×100 mL 5% aqueous lithium chloride, 2×50 mL1.0 M aqueous sodium hydroxide, and 2×100 mL brine. Separate the organicphase, dry over MgSO₄, filter, and evaporate in vacuo to yield 8.85 g(96%) of methyl 3-fluorobiphenyl-4-carboxylate as a white solid. ¹H NMR(CDCl₃, 300 MHz) 8.01 (t, J=7.8 Hz, 1H), 7.58-7.62 (m, 2H), 7.41-7.51(m, 4H), 7.37 (m, 1H), 3.96 (s, 3H); IR (cm⁻¹, KBr): 3033, 2954, 1721,1622, 1437, 1409, 1298, 1289, 1266, 1097; Anal calc'd for C₁₄H₁₁FO₂: C73.03, H 4.82; Found: C 73.06, H 4.86.

Combine methyl 3-fluorobiphenyl-4-carboxylate (8.18 g, 35.5 mmol), THF(225 mL), and 1.0 M aqueous sodium hydroxide (225 mL). Heat at 50° C.for 8 h. Cool to room temperature, add 1.0 M aqueous hydrochloric acid(300 mL), and extract with 2×200 mL ethyl acetate. Separate the organicphase, dry over MgSO₄, filter, and evaporate in vacuo to afford 7.12 g(93%) of 3-fluorobiphenyl-4-carboxylic acid as a white solid. ¹H NMR(d₆-DMSO, 300 MHz) 13.22 (br s, 1H), 7.93 (t, J=8.1 Hz, 1H), 7.74-7.78(m, 2H), 7.60-7.66 (m, 2H), 7.40-7.52 (m, 3H); IR (cm⁻¹, KBr): 3035,2666, 2575, 1699, 1621, 1563, 1408, 1298, 1265, 1193, 904; Anal calc'dfor C₁₃H₉FO₂: C 72.22, H 4.20; Found: C 72.18, H 4.35.

Combine 3-fluorobiphenyl-4-carboxylic acid 10.46 g (0.048 mol), 432 mLof methylene chloride, 7 drops of dimethylformamide, and 5.44 mL (0.062mol, 1.3 equiv) of oxalyl chloride. After 2 h, evaporate the solvent toafford 3-fluorobiphenyl-4-carbonyl chloride as a solid.

Combine 1-methylthio-1-methyl-N-(4-fluorobenzyl)-N-methylimmoniumtriflate 20.96 g (0.58 mol), 53 mL of pyridine, and1(R)-(t-butoxycarbonylamino)-2(R)-hydroxy-6-aminoindane 15.33 g (0.58mol, 1 equiv). After 4.2 h, evaporate by rotary evaporation to removemost of the pyridine, add ethyl acetate, and remove by rotaryevaporation to give a residue. Store the residue overnight at 0° C., addethyl acetate (400 mL), and extract with 200 mL of 1 N aqueous sodiumhydroxide followed by 200 mL of water. Dry the organic layer overNa₂SO₄, filter and evaporate to give a residue. Chromatograph theresidue on silica gel eluting with a gradient of 3-10% methanol inmethylene chloride to afford 18.85 g (76%) of 1(R)-(t-butoxycarbonylamino)-2(R)-hydroxy-6-(1-methyl-N′-(4-fluorobenzyl)-N′-methylamidino)indane:MS m/z=428 (M+1), mp=123-132° C.

Combine 1(R)-(t-butoxycarbonylamino)-2(R)-hydroxy-6-(1-methyl-N′-(4-fluorobenzyl)-N′-methylamidino)indane18.86 g (0.044 mol) and 110 mL of trifluoroacetic acid in an ice bath.When the addition is complete remove the ice bath and stir at roomtemperature for 2.5 h. Evaporate the reaction mixture by rotaryevaporation to afford a residue. Dissolve the residue in 100 mL ofmethylene chloride and cool in an ice bath to about 13° C., add 1Naqueous sodium hydroxide (272 mL), followed by3-fluorobiphenyl-4-carbonyl chloride 11.26 g (0.048 mol, 1.1 equiv)dissolved in 120 mL of methylene chloride. Add an additional 30 mL of 1N aqueous sodium hydroxide and stir at about 10° C. for 40 min.Partition the reaction mixture between water and methylene chloride.Separate the layers and extract the organic with water, dry, andconcentrated on a rotary evaporator to afford 20.67 g of residue.Chromatograph the residue on silica gel eluting with a gradient of 3-10%methanol in methylene chloride, followed by a second chromatography onsilica gel using a Prep 2000 eluting with a gradient of 3-10% methanolin methylene chloride, to afford 11.34 g (49%) of the title compound: MSm/z=526 (M+1).

As will be understood by one of ordinary skill in the art, an alternatename for the title compound is6-(1-((4-fluorobenzyl)methylamino)ethylideneamino)-2-hydroxy-1-(2-fluorobiphenylamido)indane.

EXAMPLE 4-1 2′,6′-Dichlorobiphenyl-4-carboxylic acid(R)-(6-(1-((4-fluorobenzyl)methylamino)ethylideneamino)-2(R)-hydroxyindan-1-yl)amide

Add DMF (1.6 mL) to a mixture ofN-cyclohexylcarbodiimide-N-methylpolystyrene resin (Novobiochem, 2.0mmol/g) (150 mg, 0.30 mmol) and 2′,6′-dichlorobiphenyl-4-carboxylic acid(14 mg, 0.05 mmol) followed by a solution of N-hydroxysuccinimide (2.3mg, 0.02 mmol) in DMF (0.2 mL) and then a solution ofN′-(3-amino-2-hydroxyindan-5-yl)-N-(4-fluorobenzyl)-N-methyl-acetamidine(6.6 mg, 0.02 mmol) in DMF (0.2 mL). Agitate the mixture for 16 hoursthen add polystyrene trisamine resin (Argonaut Technologies, 3.7 mmol/g)(100 mg, 0.37 mmol) and agitate for a further 24 hours. Filter themixture to deliver a 0.01 M solution of the title compound. MS (m/e):577 (M⁺).

Examples 4-2 through 4-37 are prepared essentially as Example 4-1.

Ex. # Compound Name MS (m/e) 4-2 2′,6′-Dichlorobiphenyl-4-carboxylicacid (R)-(6-(1-((4- 542 (M⁺)fluorobenzyl)methylamino)ethylideneamino)-2(R)-hydroxyindan- 1-yl)amide4-3 2-Methylbiphenyl-4-carboxylic acid (R)-(6-(1-((4- 522 (M⁺)fluorobenzyl)methylamino)ethylideneamino)-2(R)- hydroxyindan-1-yl)amide4-4 2′-Chlorobiphenyl-4-carboxylic acid (R)-(6-(1-((4- 542 (M⁺)fluorobenzyl)methylamino)ethylideneamino)-2(R)- hydroxyindan-1-yl)amide4-5 4′-Trifluoromethylbiphenyl-4-carboxylic acid (R)-(6-(1-((4- 576 (M⁺)fluorobenzyl)methylamino)ethylideneamino)-2(R)- hydroxyindan-1-yl)amide4-6 3-Chloro-2′,4′-difluorobiphenyl-4-carboxylic acid (R)-(6-(1- 578(M⁺) ((4-fluorobenzyl)methylamino)ethylideneamino)-2(R)-hydroxyindan-1-yl)amide 4-7 2′-Trifluoromethylbiphenyl-4-carboxylic acid(R)(6-(1-((4- 576 (M⁺) fluorobenzyl)methylamino)ethylideneamino)-2(R)-hydroxyindan-1-yl)amide 4-8 4′-Methylbiphenyl-4-carboxylic acid(R)-(6-(1-((4- 522 (M⁺) fluorobenzyl)methylamino)ethylideneamino)-2(R)-hydroxyindan-1-yl)amide 4-9 6-(2,6-Difluorophenyl)pyridine-3-carboxylicacid (R)-(6-(1- 545 (M⁺)((4-fluorobenzyl)methylamino)ethylideneamino)-2(R)-hydroxyindan-1-yl)amide 4-10 6-(2-Methylphenyl)pyridine-3-carboxylic acid(R)-(6-(1-((4- 523 (M⁺) fluorobenzyl)methylamino)ethylideneamino)-2(R)-hydroxyindan-1-yl)amide 4-11 3′,4′-Difluorobiphenyl-4-carboxylic acid(R)-(6-(1-((4- 544 (M⁺) fluorobenzyl)methylamino)ethylideneamino)-2(R)-hydroxyindan-1-yl)amide 4-12 3′,5′-Dimethylbiphenyl-4-carboxylic acid(R)-(6-(1-((4- 536 (M⁺) fluorobenzyl)methylamino)ethylideneamino)-2(R)-hydroxyindan-1-yl)amide 4-13 4-Cyclohexylphenyl-1-carboxylic acid(R)-(6-(1-((4- 514 (M⁺) fluorobenzyl)methylamino)ethylideneamino)-2(R)-hydroxyindan-1-yl)amide 4-14 3′-Cyanobiphenyl-4-carboxylic acid(R)-(6-(1-((4- 533 (M⁺) fluorobenzyl)methylamino)ethylideneamino)-2(R)-hydroxyindan-1-yl)amide 4-15 3′,5′-Difluorobiphenyl-4-carboxylic acid(R)-(6-(1-((4- 544 (M⁺)fluorobenzyl)methylamino)ethylideneamino)-2(R)-hydroxyindan- 1-yl)amide4-16 3′-Fluorobiphenyl-4-carboxylic acid (R)-(6-(1-((4- 526 (M⁺)fluorobenzyl)methylamino)ethylideneamino)-2(R)- hydroxyindan-1-yl)amide4-17 2′,4′-Difluorobiphenyl-4-carboxylic acid (R)-(6-(1-((4- 544 (M⁺)fluorobenzyl)methylamino)ethylideneamino)-2(R)- hydroxyindan-1-yl)amide4-18 2′,3′-Dichlorobiphenyl-4-carboxylic acid (R)-(6-(1-((4- 576 (M⁺)fluorobenzyl)methylamino)ethylideneamino)-2(R)- hydroxyindan-1-yl)amide4-19 4′-Chlorobiphenyl-4-carboxylic acid (R)-(6-(1-((4- 542 (M⁺)fluorobenzyl)methylamino)ethylideneamino)-2(R)- hydroxyindan-1-yl)amide4-20 3′-Chlorobiphenyl-4-carboxylic acid (R)-(6-(1-((4- 542 (M⁺)fluorobenzyl)methylamino)ethylideneamino)-2(R)- hydroxyindan-1-yl)amide4-21 4-Trifluoromethylphenyl-1-carboxylic acid (R)-(6-(1-((4- 500 (M⁺)fluorobenzyl)methylamino)ethylideneamino)-2(R)- hydroxyindan-1-yl)amide4-22 4-Methylphenyl-1-carboxylic acid (R)-(6-(1-((4- 446 (M⁺)fluorobenzyl)methylamino)ethylideneamino)-2(R)-hydroxyindan- 1-yl)amide4-23 3′,5′-Dichlorobiphenyl-4-carboxylic acid (R)-(6-(1-((4- 576 (M⁺)fluorobenzyl)methylamino)ethylideneamino)-2(R)- hydroxyindan-1-yl)amide4-24 2′,4′,6′-Trimethylbiphenyl-4-carboxylic acid (R)-(6-(1-((4- 550(M⁺) fluorobenzyl)methylamino)ethylideneamino)-2(R)-hydroxyindan-1-yl)amide 4-256-(4-fluoro-2-methylphenyl)pyridine-3-carboxylic acid (R)- 541 (M⁺)(6-(1-((4-fluorobenzyl)methylamino)ethylideneamino)-2(R)-hydroxyindan-1-yl)amide 4-265-(2,4-Difluorophenyl)pyridine-2-carboxylic acid (R)-(6-(1- 545 (M⁺)((4-fluorobenzyl)methylamino)ethylideneamino)-2(R)-hydroxyindan-1-yl)amide 4-272′-Fluoro-4′-trifluoromethylbiphenyl-4-carboxylic acid (R)- 594 (M⁺)(6-(1-((4-fluorobenzyl)methylamino)ethylideneamino)-2(R)-hydroxyindan-1-yl)amide 4-28 4-(Pyrrol-1-yl)phenyl-1-carboxylicacid (R)-(6-(1-((4- 597 (M⁺)fluorobenzyl)methylamino)ethylideneamino)-2(R)- hydroxyindan-1-yl)amide4-29 6-Methylpyridine-3-carboxylic acid (R)-(6-(1-((4- 447 (M⁺)fluorobenzyl)methylamino)ethylideneamino)-2(R)- hydroxyindan-1-yl)amide4-30 4-Cyanophenyl-1-carboxylic acid (R)-(6-(1-((4- 457 (M⁺)fluorobenzyl)methylamino)ethylideneamino)-2(R)-hydroxyindan- 1-yl)amide4-31 3,2′,6′-Trifluorobiphenyl-4-carboxylic acid (R)-(6-(1-((4- 562 (M⁺)fluorobenzyl)methylamino)ethylideneamino)-2(R)- hydroxyindan-1-yl)amide4-32 3,2′,6′-Trifluorobiphenyl-4-carboxylic acid (R)-(6-(1-((4- 541 (M⁺)fluorobenzyl)methyl)amino)ethylideneamino)-2(R)- hydroxyindan-1-yl)amide4-33 2′-Methylbiphenyl-4-carboxylic acid (R)-(6-(1-((4- 522 (M⁺)fluorobenzyl)methylamino)ethylideneamino)-2(R)- hydroxyindan-1-yl)amide4-34 2′-Methoxybiphenyl-4-carboxylic acid (R)-(6-(1-((4- 538 (MH⁺)fluorobenzyl)methylamino)ethylideneamino)-2(R)- hydroxyindan-1-yl)amide4-35 3 ,2′-Difluorobiphenyl-4-carboxylic acid (R)-(6-(1-((4- 544 (MH⁺)fluorobenzyl)methylamino)ethylideneamino)-2(R)-hydroxyindan- 1-yl)amide4-36 6′-Fluoro-2′-trifluoromethylbiphenyl-4-carboxylic acid (R)- 594(MH⁺) (6-(1-((4-fluorobenzyl)methylamino)ethylideneamino)-2(R)-hydroxyindan-1-yl)amide

EXAMPLE 5-1 6-Cyanopyridine-3-carboxylic acid(R)-(6-(1-((4-fluorobenzyl)methylamino)ethylideneamino)-2(R)-hydroxyindan-1-yl)amide

Dissolve equimolar amounts ofN′-(3-amino-2-hydroxyindan-5-yl)-N-(4-fluorobenzyl)-N-methylacetamidine(229 mg, 0.70 mmol) and 6-cyano-3-carboxypyridine (0.70 mmol, 103 mg) inanhydrous DMF (4.0 mL). Add triethylamine (0.487 mL, 3.50 mmol),followed by benzotriazol-1-yloxytris-dimethylamino phophoniumhexafluorophosphate (296 mg, 0.70 mmol). Allow the reaction to stir atroom temperature for 0.5 hours. Dilute the reaction with water (100 mL)and extract with EtOAc (3×50 mL). Dry the combined organic layers overMgSO₄, filter and concentrate. Dissolve the crude reaction product inTHF (5.0 mL). Add hydroxide resin (BIO-RAD AG 1-X8 resin, 20-50mesh—washed with water), 1 M NaOH, MeOH, ether and dried in vacuo) untilsolution is basic and stir at ambient temperature for 24 h. Filter thereaction, wash with additional THF, evaporate onto silica gel and purifyby flash column chromatography with EtOAc/Hexanes to give 253 mg (79%)of the title compound as a white solid. MS (m/e): 458 (M+).

Examples 5-2 through 5-8 are prepared essentially as Example 5-1.

Ex. # Compound Name MS (m/e) 5-2 3,5-Difluorobiphenyl-4-carboxylic acid(R)-(6-(1-((4- 544.2 (M⁺)fluorobenzyl)methylamino)ethylideneamino)-2(R)-hydroxyindan- 1-yl)amide5-3 4-(Thien-3-yl)phenyl-1-carboxylic acid (R)-(6-(1-((4- 514.1 (M⁺)fluorobenzyl)methylamino)ethylideneamino)-2(R)-hydroxyindan- 1-yl)amide5-4 6-Trifiuoromethylpyridine-3-carboxylic acid (R)-(6-(1-((4- 501.1(M⁺) fluorobenzyl)methylamino)ethylideneamino)-2(R)-hydroxyindan-1-yl)amide 5-5 3-Fluoro-2′-trifluoromethylbiphenyl-4-carboxylic acid(R)- 594.1 (M⁺)(6-(1-((4-fluorobenzyl)methylamino)ethylideneamino)-2(R)-hydroxyindan-1-yl)amide 5-6 2-Fluoro-4-trifluoromethylphenyl-1-carboxylic acid(R)-(6- 518.4 (M⁺)(1-((4-fluorobenzyl)methylamino)ethylideneamino)-2(R)-hydroxyindan-1-yl)amide 5-7 2′-Chloro-3-fluorobiphenyl-4-carboxylic acid(R)-(6-(1-((4- 560.2 (M⁺)fluorobenzyl)methylamino)ethylideneamino)-2(R)-hydroxyindan- 1-yl)amide5-8 5-Phenylpyrazine-2-carboxylic acid (R)-(6-(1-((4- 510.3 (M⁺)fluorobenzyl)methylamino)ethylideneamino)-2(R)-hydroxyindan- 1-yl)amide

EXAMPLE 6-1 6-(2-Chlorophenyl)pyridine-3-carboxylic acid(R)-(6-(1-((4-fluorobenzyl)methylamino)ethylideneamino)-2(R)-hydroxyindan-1-yl)amide

DissolveN′-(3(R)-amino-2(R)-hydroxyindan-5—yl)-N-(4-fluorobenzyl)-N-methylacetamidine(42 mg, 0.13 mmol) and 6-(2-chlorophenyl)pyridine-3-carboxylic acid (103mg, 0.70 mmol) in anhydrous DMF (2.5 mL). Add triethylamine (0.178 mL)followed by benzotriazol-1-yloxytris-dimethylamino phophoniumhexafluorophosphate (54 mg, 0.13 mmol). Allow the reaction to stir atroom temperature until completion. Dilute the reaction with water (100mL) and extract with EtOAc. Dry the combined organic layers over MgSO₄,filter, and concentrate. Purify by flash column chromatography withCHCl₃/MeOH mixtures to yield 276 mg (35.7%) of solid title compound isisolated. MS (m/e): 543.0 (M⁺).

Examples 6-2 through 6-4 are prepared essentially as Example 6-1.

Ex. # Compound Name MS (m/e) 6-26-(2,4-Difluorophenyl)pyridine-3-carboxylic acid (R)-(6-(1- 545.1 (M⁺)((4-fluorobenzyl)methylamino)ethylideneamino)-2(R)-hydroxyindan-1-yl)amide 6-3 4-Iodophenyl-1-carboxylic acid (R)-(6-(1-((4- 558.0 (M⁺)fluorobenzyl)methylamino)ethylideneamino)-2(R)-hydroxyindan- 1-yl)amide6-4 6-(Thien-3-yl)pyridine-3-carboxylic acid (R)-(6-(1-((4- 515.4 (MH⁺)fluorobenzyl)methylamino)ethylideneamino)-2(R)-hydroxyindan- 1-yl)amide

EXAMPLE 7-1 4-(Pyridin-3-yl)phenyl-1-carboxylic acid(R)-(6-(1-((4-fluorobenzyl)methylamino)ethylideneamino)-2(R)-hydroxyindan-1-yl)amide

Combine 4-iodophenyl-1-carboxylic acid(R)-(6-(1-((4-fluorobenzyl)methylamino)ethylideneamino)-2(R)-hydroxyindan-1-yl)amide(0.131 g, 0.235 mmol), 2-(tributylstannyl)pyridine (0.129 g, 0.352 mmol)and tetrakis(triphenylphosphine)palladium(0) (0.406 g, 0.352 mmol) indioxane at 80° C. Heat with stirring until completion. Remove thesolvent by evaporation. Dilute the residue with Ethyl acetate and stirwith an equal volume of saturated potassium fluoride for 3 hours. Filterthe solution through a pad of celite. Separate the organic layer fromthe aqueous layer and dry over magnesium sulfate. Remove the organicsolvent by evaporation and purify via flash chromatography withdichloromethane and methanol to yield 0.035 g (30%) of the titlecompound as solid material. MS (m/e): 509.2 (M+).

EXAMPLE 8-1 2′,4′,6′-Trifluorobiphenyl-4-carboxylic acid(R)-(6-(1-((4-fluorobenzyl)methylamino)ethylideneamino)-2(R)-hydroxyindan-1-yl)amide

Combine 2′,4′,6′-trifluorobiphenyl-4-carboxylic acid (66 mg, 0.26 mmol),EDC (53 mg, 0.28 mmol) and N-hydroxysuccinimide (0.33 mg, 0.28 mmol) indichloromethane and stir until completion. Wash the solution with 1 Nhydrochloric acid. The organic layer is dried over magnesium sulfate andconcentrated. The residue is combined withN′-(3(R)-amino-2(R)-hydroxyindan-5-yl)-N-(4-fluorobenzyl)-N-methylacetamidine(42 mg, 0.13 mmol) in dichloromethane and stirred until completion ofreaction. The solvent is removed by evaporation and the residue purifiedvia flash chromatography with dichloromethane and methanol to yield 37mg of the title compound. MS (m/e): 562.0 (M⁺).

EXAMPLE 9-1 3,4′-Difluorobiphenyl-4-carboxylic acid(R)-(6-(1-((4-fluorobenzyl)methylamino)ethylideneamino)-2(R)-hydroxyindan-1-yl)amide

Stir a mixture of 3,4′-difluorobiphenyl-4-carboxylic acid (160 mg, 0.684mmol), N-hydroxysuccinimmide (79 mg, 0.684 mmol) and DCC (141 mg, 0.684mmol) in 15 mL of methylene chloride at rt for 2 h. Combine(6-(1-((4-fluorobenzyl)methylamino)ethylideneamino)-2(R)-hydroxyindan-1-yl)carbamicacid tert-butyl ester (256 mg. 0.62 mmol) with TFA (2 mL) at 0° C. andstir for 2 hr. Evaporate TFA under reduced pressure. Dissolve theresidue in methylene chloride and evaporate to dryness-repeat thisprocess three times. Add 5 mL of methylene chloride and 1 mL oftriethylamine. Add this resulting solution to the above mixture and stirat rt for 12 h.

Pour the mixture into methylene chloride, wash with water, dry withNa₂SO₄, and concentrate. Purify the residue by column chromatography(silica gel, 3% MeOH in CH₂Cl₂) to give 199 mg of the title compound asa white solid (55% yield).

¹H NMR (CDCl3) δ 8.21 (1H, t, J=8.4 Hz), 7.58 (2H, dd, J=8.8 and 4.8Hz), 7.32 (1H, d, J=13.2 Hz), 7.26-7.23 (2H, m), 7.19-7.12 (3H, m), 7.04(2H, t, J=8.8 Hz), 6.68 (1H, s), 6.66 (1H, s), 5.32 (1H, t, J=5.2 Hz),4.82-4.72 (1H, m), 4.64 (2H, s), 4.56(1H, q, J=6.4 Hz), 3.32(1H, dd,J=15.6 and 8.0 Hz), 3.00(3H, s), 2.96(1H, dd, J=15.2 and 8.4 Hz), 1.96(3H, s). MS 544 (MH⁺).

Examples 9-2 through 9-6 are prepared essentially as Example 9-1.

Ex. # Compound Name MS (m/e) 9-2 4′-Fluorobiphenyl-4-carboxylic acid(R)-(6-(1-((4- 526 (MH⁺) fluorobenzyl)methylamino)ethylideneamino)-2(R)-hydroxyindan-1-yl)amide 9-3 4′-Fluoro-2′-methoxybiphenyl-4-carboxylic556 (MH⁺) acid (R)-(6-(1-((4- fluorobenzyl)methylamino)ethylideneamino)-2(R)-hydroxyindan-1-yl)amide 9-4 2′,6′-Difluorobiphenyl-4-carboxylicacid (R)-(6-(1- 544 (MH⁺) ((4-fluorobenzyl)methylamino)ethylideneamino)-2(R)-hydroxyindan-1-yl)amide 9-5 2′-Fluorobiphenyl-4-carboxylic acid(R)-(6-(1-((4- 526.1 (MH⁺)  fluorobenzyl)methylamino)ethylideneamino)-2(R)- hydroxyindan-1-yl)amide9-6 3,2′,4′-Trifluorobiphenyl-4-carboxylic acid (R)-(6-   562 (M + 1)(1-((4-fluorobenzyl)methylamino)ethylideneamino)-2(R)-hydroxyindan-1-yl)amide

EXAMPLE 10-1 4-Bromophenyl-1-carboxylic acid(R)-(6-(1-((furan-2-ylmethyl)methylamino)ethylideneamino)-2(R)-hydroxyindan-1-yl)amide

To a mixture of thioacetamide (10.04 g, 133.64 mmol) and K₂CO₃ (45.80 g,331.38 mmol) in 100 ml THF at 0° C. add phthaloyl chloride (28.49 g,140.32 mmol) dropwise. Raise the reaction temperature to 25° C. after 2hours and allow it to stir for an additional 2 hours before cooling thereaction mixture to 0° C. again. Quench the reaction by adding 125 mL ofice water dropwise. Extract the reaction mixture with EtOAc (2×). Drythe organic layer with MgSO₄ and remove the solvent in vacuo to yield3.7 g of N-thioacetyl-isoindole-1,3-dione as a crude reddish solid.

Dissolve N-methylfurfurylamine (117.2 mg, 1.05 mmol) in 20 ml Et₂O at25° C. To this add N-thioacetyl-isoindole-1,3-dione (294.4 mg, 1.43mmol) and allow to stir for 24 hours. Add MeOTf(181.7 mg, 1.11 mmol) tothe reaction mixture and allow to stir for an additional 23 hours.Decant the Et₂O from the oil and triturate the oil with Et₂O-repeat thisthree times. Remove any excess Et₂O from the oily residue in vacuo toobtain 320.4 mg of crude thioimidate as an oil. Dissolve this crudeproduct (161.1 mg, 0.483 mmol) in 10 mL pyridine and addN-(6-amino-2-hydroxyindan-1-yl)-4-bromobenzamide (107.5 mg, 0.310 mmol).Allow the reaction to stir at 25° C. for 22 hours. Remove the solvent invacuo and partition the residue between CH₂Cl₂ and saturated aqueousNaHCO₃. Dry the organic layer with MgSO₄. Filter and remove the solventin vacuo to give 146.2 mg of crude product. Purify via Biotagechromatography (1% MeOH/EtOAc) to afford 63.6 mg of the title compoundas an off-white solid (43%). MS (m/e): 483 (M+1).

EXAMPLE 11-1 4-Bromophenyl-1-carboxylic acid(R)-(6-(1-((4-fluorobenzyl)amino)ethylideneamino)-2(R)-hydroxyindan-1-yl)amide

Step a) Combine (6-amino-2-hydroxyindan-1-yl)carbamic acid tert-butylester (3.7 g, 14.0 mmol) with 10 mL of TFA at 0° C. Stir the mixture for1 h and evaporate to dryness. Add to the residue 6.5 mL of triethylamineand 30 mL of methylene chloride. Combine this mixture slowly with asolution of 4-bromobenzoic acid benzotriazole-1-yl ester in 15 mL ofmethylene chloride at 0° C. Stir the resulting mixture for 12 h at rt.Filter to give 3.4 g (70% yield) ofN-(6-amino-2-hydroxyindan-1-yl)-4-bromobenzamide as white solid. MS 348(MH⁺).

Step b) Add acetyl chloride (9.28 g, 118 mmol) to a mixture oftriethylamine (16.0 g, 158 mmol) and 4-fluorobenzylamine (9.90 g, 78.8mmol) in 200 mL of ethyl acetate at 0° C. and stir for 12 hours. Add 100mL of water to the mixture. Extract aqueous layer with ethyl acetate(3×100 mL). Combine organic layers and wash with brine then dry overanhydrous Na₂SO₄ Remove solvent under reduced pressure to giveN-(4-fluorobenzyl)acetamide (13.5 gm) in 100% yield as a yellow oil. AddNaH (6.5 g, 162 mmol) to N-(4-fluorobenzyl)-acetamide (13.5 g, 80.8mmol) in 200 mL of tetrahydrofuran at 0° C. and stir for 4 hours. Thenadd methyl iodide (22.9 g, 161.6 mmol) to the above mixture and stir for12 hours. Pour mixture into 200 mL of water. Extract aqueous layer withmethylene chloride (3×200 mL). Combine organic layers and wash withbrine then dry over anhydrous Na₂SO₄. Remove solvent under reducedpressure. Purify the residue by column chromatography (silica gel, 5%acetone in hexanes, 50% acetone in hexanes) to giveN-(4-fluorobenzyl)-N-methylacetamide (9.1 gm) in 64% yield as a yellowoil. Add Lawesson reagent (20.3 g, 50.3 mmol) toN-(4-fluorobenzyl)-N-methylacetamide (9.1 g, 50.3 mmol) in 200 mL oftoluene and heat to 100° C. for three hours. Remove solvent underreduced pressure. Purify the residue with a column chromatography(silica gel, 2% methylene chloride in hexanes, 100% methylene chloride)to give N-(4-fluorobenzyl)-N-methylthioacetamide (5.6 g) 56.5% as ayellow solid: MS 198 (MH⁺).

Step c) Add Lawesson reagent (1.8 g, 3.6 mmol) toN-(4-fluorobenzyl)acetamide (0.9 g, 5.4 mmol) in 50 mL of toluene andheat to 80° C. for 12 hours. Remove solvent under reduced pressure.Purify the residue by column chromatography (silica gel, Acetone:Hexane=20:80) to give a mixture. Wash with ether and discard insolublesolid impurities by filtration. Remove solvent under reduced pressure togive N-(4-fluorobenzyl)-thioacetamide (0.7 gm) in 71% as a yellow solid:MS 184 (MH⁺).

Step d) Add methyl trifluoromethanesulfonate (0.190 g, 1.16 mmol) to asolution of N-(3-fluorobenzyl)thioacetamide (0.106 g, 0.580 mmol) in 10mL of CH₂Cl₂ at room temperature. Stir the mixture for 30 minutes andremove the solvent under reduced pressure. Dissolve the residue in 5 mLof pyridine. Then add the trifluoroacetic acid salt ofN-(6-amino-2-hydroxyindan-1-yl)-4-bromobenzamide to the solution. Stirfor three hours. Remove pyridine under reduced pressure. Purify theresidue with column chromatography (silica gel/Hexanes:Acetone =7:3,1:1) to give 40 mg of the title compound in 14% yield as a white solid:MS 496(MH⁺).

Examples 11-2 through 11-17 are prepared essentially as Example 11-1.

Ex. # Compound Name MS (m/e) 11-2 4-Bromophenyl-1-carboxylic acid(R)-(6-(1-((3- 496 (MH⁺) fluorobenzyl)amino)ethylideneamino)-2(R)-hydroxyindan-1-yl)amide 11-3 4-Bromophenyl-1-carboxylic acid(R)-(6-(1-((1,3- 522 (MH⁺)benzodioxol-5-ylmethyl)amino)ethylideneamino)-2(R)-hydroxyindan-1-yl)amide 11-4 4-Bromophenyl-1-carboxylic acid(R)-(6-(1-((4- 508 (MH⁺) methoxybenzyl)amino)ethylideneamino)-2(R)-hydroxyindan-1-yl)amide 11-5 4-Bromophenyl-1-carboxylic acid(R)-(6-(1-((3- 508 (MH⁺) methoxybenzyl)amino)ethylideneamino)-2(R)-hydroxyindan-1-yl)amide 11-6 4-Bromophenyl-1-carboxylic acid(R)-(6-(1-((2- 508 (MH⁺) methoxybenzyl)amino)ethylideneamino)-2(R)-hydroxyindan-1-yl)amide 11-7 4-Bromophenyl-1-carboxylic acid(R)-(6-(1-((4- 510 (MH⁺) fluorobenzyl)methylamino)ethylideneamino)-2(R)-hydroxyindan-1-yl)amide 11-8 4-Bromophenyl-1-carboxylic acid(R)-(6-(1-((2-(4- 510 (MH⁺)fluorophenyl)ethyl)amino)ethylideneamino)-2(R)- hydroxyindan-1-yl)amide11-9 4-Bromophenyl-1-carboxylic acid (R)-(6-(1-((2-(2- 510 (MH⁺)fluorophenyl)ethyl)amino)ethylideneamino)-2(R)- hydroxyindan-1-yl)amide11-10 4-Bromophenyl-1-carboxylic acid (R)-(6-(1-((2-(4- MSmethoxyphenyl)ethyl)amino)ethylideneamino)-2(R)- 522 (MH⁺)hydroxyindan-1-yl)amide 11-11 4-Bromophenyl-1-carboxylic acid(R)-(6-(1-((2-(3- 510 (MH⁺)fluorophenyl)ethyl)amino)ethylideneamino)-2(R)- hydroxyindan-1-yl)amide11-12 4-Bromophenyl-1-carboxylic acid (R)-(6-(1-((2-(2- 522 (MH⁺)methoxyphenyl)ethyl)amino)ethylideneamino)-2(R)- hydroxyindan-1-yl)amide11-13 4-Bromophenyl-1-carboxylic acid (R)-(6-(1-((2-(3- 522 (MH⁺)methoxyphenyl)ethyl)amino)ethylideneamino)-2(R)- hydroxyindan-1-yl)amide11-14 4-Bromophenyl-1-carboxylic acid (R)-(6-(1-((2-(2,3- 552 (MH⁺)dimethoxyphenyl)ethyl)amino)ethylideneamino)-2(R)-hydroxyindan-1-yl)amide 11-15 4-Bromophenyl-1-carboxylic acid(R)-(6-(1-((2-(1,3- 536 (MH⁺)benzodioxol-5-yl)ethyl)amino)ethylideneamino)-2(R)-hydroxyindan-1-yl)amide 11-16 4-Bromophenyl-1-carboxylic acid(R)-(6-(1-((2-(4- 524 (MH⁺)fluorophenyl)ethyl)amino)ethylideneamino)-2(R)- hydroxyindan-1-yl)amide11-17 4-Bromophenyl-1-carboxylic acid (R)-(6-(1-((2-(2- 536 (MH⁺)methoxyphenyl)ethyl)amino)ethylideneamino)-2(R)- hydroxyindan-1-yl)amide

EXAMPLE 12-1 Biphenyl-4-carboxylic acid(R)-(6-(1-((4-fluorobenzyl)methylamino)ethylideneamino)-2(R)-hydroxyindan-1-yl)amide

Combine (6-amino-2-hydroxyindan-1-yl)carbamic acid tert-butyl ester (1.5g, 5.68 mmol) with 5 mL of TFA at 0° C. Stir the mixture for 1 h andthen evaporate to dryness. Added 3.0 mL of triethylamine and 30 mL ofmethylene chloride the residue. To this mixture, add a solution ofbiphenyl-4-carboxylic acid 2,5-dioxo-pyrrolidin-1-yl ester (1.76 g, 5.96mmol) in 15 mL of methylene chloride. Stir the resulting mixture for 12h. Evaporate solvent and purify the residue by a column chromatography(silica gel/MeOH:CH₂Cl₂=9:1) to give 1.88 g (96% yield) ofbiphenyl-4-carboxylic acid (6-amino-2-hydroxyindan-1-yl)amide; MS 345(MH⁺).

Add methyl trifluoromethanesulfonate (0.290 g, 1.76 mmol) to a solutionof N-(4-fluorobenzyl)thioacetamide (0.2 gm, 1.0 mmol) in 5 mL of CH₂Cl₂at room temperature. Stir the mixture for 30 minutes and remove thesolvent under reduced pressure. Dissolve the residue in 5 mL ofpyridine. Then add biphenyl-4-carboxylic acid(6-amino-2-hydroxyindan-1-yl)amide to the solution. Stir for 12 hours.Remove pyridine under reduced pressure. Purify the residue with columnchromatography (silica gel/Hexanes: Acetone =7:3, 1:1) to give 78 mg ofthe title compound in 35% yield as a white solid: ¹H NMR (DMSO-d₆) δ8.78 (1H, d, J=8.8 Hz), 8.04 (2H, d, J=8.8 Hz), 7.79 (2H, d, J=8.4 Hz),7.74 (2H, dd, J=7.4, 1.6 Hz), 7.50 (2H, t, J=8.0 Hz), 7.41 (1H, t, J=7.2Hz), 7.28 (2H, dd, J=8.8, 6.6 Hz), 7.14 (2H, t, J=8.8 Hz), 7.04 (1H, t,J=7.6 Hz), 6.78 (1H, d, J=7.2 Hz), 6.37 (1H, s), 5.33 (1H, d, J=5.6 Hz),5.27 (1H, t, J=7.6 Hz), 4.59 (2H, s), 4.45 (1H, q, J=6.0 Hz), 3.10 (1H,dd, J=14.8, 7.2 Hz), 2.90 (3H, s), 2.68 (1H, dd, J=14.8, 7.2 Hz), 1.88(3H, s); MS 508 (MH⁺).

Examples 12-2 through 12-7 are prepared essentially as Example 12-1.

Ex. # Compound Name MS (m/e) 12-2 Biphenyl-4-carboxylic acid(R)-(6-(1-((3- 520 (MH⁺) methoxybenzyl)methylamino)ethylideneamino)-2(R)-hydroxyindan-1-yl)-amide 12-3 Biphenyl-4-carboxylic acid(R)-(6-(1-((3,4- 526 (MH⁺)difluorobenzyl)methylamino)ethylideneamino)-2(R)-hydroxyindan-1-yl)amide 12-4 Biphenyl-4-carboxylic acid(R)-(6-(1-((2-(4- 508 (MH⁺)fluorophenyl)ethyl)amino)ethylideneamino)-2(R)- 12-5Biphenyl-4-carboxylic acid (R)-(6-(1-((2-(2- 520 (MH⁺)methoxyphenyl)ethyl)amino)ethylideneamino)-2(R)- hydroxyindan-1-yl)amide12-6 Biphenyl-4-carboxylic acid (R)-(6-(1-((2-(2- 508 (MH⁺)fluorophenyl)ethyl)amino)ethylideneamino)-2(R)- hydroxyindan-1-yl)amide12-7 Biphenyl-4-carboxylic acid (R)-(6-(1-((2-(4- 522 (MH⁺)fluorophenyl)ethyl)methylamino)ethylideneamino)-2(R)-hydroxyindan-1-yl)amide

EXAMPLE 13-1 Biphenyl-4-carboxylic acid(R)-(7-(1-((4-fluorobenzyl)methylamino)ethylideneamino)-2(R)-hydroxy-1,2,3,4-tetrahydronaphthalen-1-yl)amide

Stir a mixture of 1-amino-7-nitro-1,2,3,4-tetrahydronaphthalen-2-ol (500mg, 2.40 mmol) and 1N NaOH (4.8 mL, 4.8 mmol) in a mixture of 50 mL oftoluene and 50 mL of water for 30 minutes. Added biphenyl-4-carbonylchloride (570 mg, 2.64 mmol) slowly and stir for four hours at roomtemperature. Remove the solid by filtration, and wash with toluene.Evaporate solvent under reduced pressure. Biphenyl-4-carboxylic acid(2-hydroxy-7-nitro-1,2,3,4-tetrahydro-naphthalen-1-yl)-amide is obtainedin 54% as a white solid: ¹H NMR (DMSO-d₆) δ 8.87 (1H, d, J=8.4 Hz), 8.03(2H, d, J=8.4 Hz), 7.99 (2H, d, J=7.6 Hz), 7.79 (2H, d, J=8.4 Hz), 7.74(2H, d, J=8.4 Hz), 7.50 (2H, t, J=7.2 Hz), 7.43(2H, t, J=6.8 Hz), 5.22(1H, d, J=4.4 Hz), 5.11 (1H, t, J=7.6 Hz), 3.95-4.01 (1H, m),2.87-3,07(2H, m), 2.08-2.15 (1H, m), 1.81-1.94 (1H, m); MS 389(MH⁺). Theabove compound (0.25 g, 0.64 mmol) is reduced in 10 mL of DMF with 48 mgof 10% Pd-C at room temperature under 60 psi (414 kPa) for overnight toform biphenyl-4-carboxylic acid(7-amino-2-hydroxy-1,2,3,4-tetrahydro-naphthalen-1-yl)-amide, obtainedin 96% as a yellow oil. MS 359 (MH⁺).

Add methyl trifluoromethanesulfonate (0.150 g, 0.915 mmol) to a solutionof N-4-fluorobenzyl-N-methylthioacetamide (0.145 g, 0.736 mmol) in 10 mLof CH₂Cl₂ at room temperature. Stir the mixture for 30 minutes andremove the solvent under reduced pressure. Then addbiphenyl-4-carboxylic acid(7-amino-2-hydroxy-1,2,3,4-tetrahydro-naphthalen-1-yl)-amide (0.22 g,0.61 mmol) followed by 5 mL of pyridine. Stir the resulting mixture for12 h. After evaporate pyridine, purify the residue by columnchromatography (silica gel, 5% MeOH in CH₂Cl₂) to give 10 mg of thetitle compound in 2.6% yield as a light yellow solid: ¹H NMR (CDCl₃) δ7.89 (2H, d, J=8.0 Hz), 8.66 (2H, d, J=8.4 Hz), 7.61 (2H, d, J=7.2 Hz),7.47 (2H, t, J=7.6 Hz), 7.39 (1H, t, J=7.6 Hz), 7.21 (1H, dd, J=6.0, 8.8Hz), 6.98-7.05 (3 B, m), 6.72 (1H, s), 6.62 (1H, dd, J=2.0, 8.4 Hz),6.56 (1H, d, J=7.2 Hz), 5.22 (1H, t, J=7.6 Hz), 4.60 (2H, s), 3.98-4.04(2H, m), 3.41 (11H, t, J=6.0 Hz), 2.96 (3H, s), 2.85 (2H, t, J=4.8 Hz),2.15-2.21 (2H, m), 1.87-1.95 (1H, m), 1.92 (3H, s), 1.60-1.64 (1H, m);MS 522 (MH⁺).

EXAMPLE 14-1 3-Fluorobiphenyl-4-carboxylic acid(R)-(6-(1-((3,4-difluorobenzyl)methylamino)ethylideneamino)-2(R)-hydroxyindan-1-yl)amide

Beginning with 3,4-difluorobenzylamine,N-(3,4-difluorobenzyl)-N-methyl-thioacetamide is prepared essentially asStep b, Example 11-1, and is obtained in 66% yield as a yellow solid: MS216 (MH⁺).

Add methyl trifluoromethanesulfonate (0.750 g, 3.49 mmol) to a solutionof N-(3,4-difluorobenzyl)-N-methylthioacetamide (0.145 g, 0.736 mmol) in10 mL of CH₂Cl₂ at room temperature. Stir the mixture for 30 minutes andremove the solvent under reduced pressure. Then add(6-amino-2-hydroxyindan-1-yl)-carbamic acid t-butyl ester (0.78 g, 2.97mmol) followed by 5 mL of pyridine. The resulting mixture is stirred for12 h. After pyridine is evaporated, the residue is purified by columnchromatograph (silica gel, 3% MeOH in CH₂Cl₂) to give 0.98 g of(6-(1-((3,4-difluorobenzyl)-methyl-amino)-ethylideneamino)-2-hydroxyindan-1-yl)-carbamicacid tert-butyl ester as a white solid: MS 446 (MH⁺).

Add 5 mL of trifluoroacetic acid to(R)-(6-(1-((3,4-difluorobenzyl)methylamino)ethylideneamino)-2(R)-hydroxyindan-1-yl)carbamicacid tert-butyl ester (180 mg, 4.04 mmol) and stir for 30 minutes.Remove solvent under reduced pressure. Dissolve the residue in 15 mL ofCH₂Cl₂. Then add 3-fluorobiphenyl-4-carboxylic acid2,5-dioxopyrrolidin-1-yl ester (150 mg, 4.79 mmol) and triethylamine(0.6 mL, 4.0 mmol) to the solution and stir for 12 hours. Pour themixture into methylene chloride, wash with water, dry with Na₂SO₄, andthen concentrate. Purify the residue by column chromatography (silicagel, 3% MeOH in CH₂Cl₂) to give 35 mg of the title compound as a whitesolid in 16% yield: ¹H NMR (CD₃OD) δ 7.88 (2H, t, J=8.0 Hz), 7.7 (2H, d,J=8.4 Hz), 7.60 (2H, dd, J=8.0, 1.2 Hz), 7.47-7.55 (3H, m), 7.43-7.47(1H, m), 7.21-7.32 (2H, m), 7.18 (1H, d, J=8.0 Hz), 7.11-7.14 (1H, m),6.71 (2H, dd, J=10.0, 1.2 Hz), 5.47 (1H, d, J=6.8 Hz), 4.69 (2H, s),4.54 (1H, q, J=7.2 Hz), 4.54 (1H, q, J=6.4 Hz), 3.29 (1H, q, J=7.2 Hz),3.07 (3H, s), 2.88 (1H, q, J=7.8 Hz), 1.99 (3H, s); MS 544 (MH⁺).

EXAMPLE 15-1 2′-Trifluoromethylbiphenyl-4-carboxylic acid(R)-(6-(1-((4-fluorobenzyl)methylamino)ethylideneamino)indan-1-yl)amide

Combine 1-aminoindan (1.0 g, 8.1 mmol) with 6.6 mL of fuming HNO₃ at−10° C. Stir the resulting mixture for 30 min, pour into ice and thenstir for 30 min. Collect the solid, wash with water, and dry to afford0.614 g (42%) of product. Mix with di-tert-butyldicarbonate (0.929 g,4.26 mmol) in THF. Combine the mixture with saturated K₂CO₃ aqueoussolution to adjust to pH11. Stir for 8 h, pour into CH₂Cl₂, wash withwater, dry, and concentrate. Dissolve the residue into a 1:1 mixture ofether and hexane, then keep at −10° C. overnight. Collect the solid anddry to give 0.57 g (59%) of (6-Nitro-indan-1-yl)carbamic acid tert-butylester. ¹H NMR (CDCl₃) d 8.16 (1H, br s), 8.11 (1H, dd, J=8.4 and 2.0Hz), 7.34 (1H, d, J=8.4 Hz), 5.25 (1H, q, J=8.8 Hz), 4.78 (1 h, br d,J=6.4 Hz), 3.08-2.00 (1H, m), 2.96-2.78 (1H, m), 2.72-2.62 (1H, m),1.93-1.83 (1H, m), 1.51 (9H, s).

Add KBH₄ (1.59 g, 29.4 mmol) slowly to a mixture of(6-nitro-indan-1-yl)carbamic acid tert-butyl ester (1.17 g, 4.2 mmol)and CuCl (1.25 g, 12.6 mmol) in 50 mL of methanol at 0° C. over 15 min.Stir the mixture for 1 h and then pass through a Florisil pad. Evaporatethe THF and dissolve the solid in EtOAc. Wash the mixture with water,dry, and concentrate to afford 0.76 g (73%) of(6-aminoindan-1-yl)carbamic acid tert-butyl ester. ¹H NMR (CDCl₃) d 6.99(1H, d, J=8.0 Hz), 6.65 (1H, s), 6.56 (1H, dd, J=8.0 and 2.0 Hz), 5.09(1H, q, J=8.0 Hz), 4.70 (1H, br d, J=7.2 Hz), 3.60 (2H, s), 2.86-2.79(1H, m), 2.75-2.67 (1H, m), 2.58-2.47 (1H, m), 1.78-1.69 (1H, m), 1.48(3H, s). 2.86-2.79 (1H, m), 2.75-2.67 (1H, m), 2.58-2.47 (1H, m),1.78-1.69 (1H, m), 1.48 (3H, s).

Add MeI (0.623 g, 4.4 mmol) to a solution ofN-4-fluorobenzyl-N-methylthioacetamide (0.662 g, 3.4 mmol) in 10 mL ofether at rt. Stir the mixture for 3 h and evaporate the ether undervacuum. Add (6-aminoindan-1-yl)carbamic acid tert-butyl ester (0.7 g,2.28 mmol) followed by 5 mL of pyridine. Stir the resulting mixture for12 h. Evaporate pyridine and purify the residue by column chromatography(silica gel, 3% MeOH in CH₂Cl₂) to give 1.26 g of(6-(1-((4-fluorobenzyl)methylamino)ethylideneamino)indan-1-yl)carbamicacid tert-butyl ester as a light yellow solid; MS 412 (MH⁺); ¹H NMR(CD₃OD) d 7.35-7.29 (2H, m), 7.18-7.10 (3H, m), 6.70 (1H, s), 6.65 (1H,d, J=7.6 Hz), 5.06 (1H, t, J=6.8 Hz), 4.69 (2H, s), 3.05 (3H, s),2.97-2.91 (1H, m), 2.84-2.77 (1H, m), 2.55-2.48 (1H, m), 1.97 (3H, s),1.52 (9H, s).

Stir a mixture of 2′-trifluoromethylbiphenyl-4-carboxylic acid (100 mg,0.376 mmol), N-hydroxysuccinimmide (43 mg, 0.376 mmol) and DCC (77 mg,0.376 mmol) in 15 mL of methylene chloride at rt for 2 h. Combine(6-(1-((4-fluorobenzyl)methylamino)ethylideneamino)indan-1-yl)carbamicacid tert-butyl ester (129 mg. 0.313 mmol) with TFA (2 mL) at 0° C. andstir for 2 hr. Evaporate TFA under reduced pressure. Dissolve theresidue in methylene chloride and evaporate to dryness; repeat thisthree times. Add 5 mL of methylene chloride and 1 mL of triethylamine.Add this resulting solution to the above mixture and stir at rt for 12h. Pour the mixture into methylene chloride, wash with water, dry withNa₂SO₄, and concentrate. Purify the residue by column chromatography(silica gel, 3% MeOH in CH₂Cl₂) to give 82 mg of the title compound as awhite solid (47% yield).

MS 560 (MH⁺); ¹H NMR (CDCl₃) d 7.83 (2H, d, J=8.0 Hz), 7.75 (1H, d, =7.6Hz), 7.56 (1H, t, J=7.2 Hz), 7.49 (1H, t, J=8.0 Hz), 7.40 (2H, d, J=8.0Hz), 7.31 (1 H, d, J=8.0 Hz), 7.26-7.21 (3H, m), 7.15 (1H, d, J=7.6 Hz),7.02 (2H, t, J=8.8 Hz), 6.75 (1H, s), 6.64 (1H, d, J=7.2 Hz), 5.67 (1H,q, J=7.6 Hz), 4.61 (2H, q, J=10.0 Hz), 2.97 (3H, s), 2.94-2.83 (1H, m),2.75-2.68 (1H, m), 2.00-1.70 (4H, br s), 1.69-1.58 (1H, m).

Examples 15-2 through 15-7 are prepared essentially as Example 15-1.

Ex. # Compound Name MS (m/e) 15-2 3,5-Difluorobiphenyl-4- 528 (MH⁺)carboxylic acid (R)-(6-(1-((4-fluorobenzyl)methylamino)ethylideneamino)indan- 1-yl)amide 15-32′-Fluorobiphenyl-4-carboxylic acid (R)-(6-(1-((4- 510 (MH⁺)fluorobenzyl)methylamino)ethylideneamino)indan- 1-yl)amide 15-42′,6′-Difluoromethylbiphenyl-4- 528 (MH⁺) carboxylic acid (R)-(6-(1-((4-fluorobenzyl)methylamino)ethylideneamino)indan- 1-yl)amide 15-5Biphenyl-4-carboxylic acid (R)-(6-(1-((4- 492 (MH⁺)fluorobenzyl)methylamino)ethylideneamino)indan- 1-yl)amide 15-63-Fluorobiphenyl-4-carboxylic acid (R)-(6-(1-((4- 510 (MH⁺)fluorobenzyl)methylamino)ethylideneamino)indan- 1-yl)amide 15-73,2′-Difluorobiphenyl-4- 528 (MH⁺) carboxylic acid (R)-(6-(1-((4-fluorobenzyl)methylamino)ethylideneamino)indan- 1-yl)amide

Example P-1 Biphenyl-4-carboxylic acid(R)-(6-(1-((4-fluorobenzyl)methylamino)ethylideneamino)-2(R)-hydroxyindan-1-yl)amidehemihydrate

Add 21.8 L of methanol to 2.86 kg of biphenyl-4-carboxylic acid(R)-(6-(1-((4-fluorobenzyl)methylamino)ethylideneamino)-2(R)-hydroxyindan-1-yl)amidesolvate. Pass the solution through a carbon impregnated filter and rinsethe filter with 24 L of methanol. Add 5.7 kg of water to the solutionover 35 min followed by 15 g of Biphenyl-4-carboxylic acid(R)-(6-(1-((4-fluorobenzyl)methylamino)ethylideneamino)-2(R)-hydroxyindan-1-yl)amidehemihydrate seed crystals. After 20 min, add 1.15 kg of water followedby 15 g of seed crystals. After 1 h, add another 1.15 kg of water over30 min followed by 15 g of seed crystals. After 10 min, add 3.4 kg ofwater over 1 h and stir the slurry at room temperature for 1 h and at 0°C. for 45 min. Collect the solid by filtration, rinse with a coldsolution of 11.4 L of methanol and 2.9 L of water, and dry to afford2.19 kg of the title compound as a white solid.

Example P-2 Biphenyl-4-carboxylic acid(R)-(6-(1-((4-fluorobenzyl)methylamino)ethylideneamino)-2(R)-hydroxyindan-1-yl)amidehemihydrate

Dissolve biphenyl-4-carboxylic acid(R)-(6-(1-((4-fluorobenzyl)methylamino)ethylideneamino)-2(R)-hydroxyindan-1-yl)amidesolvate (2.0 g) in methanol (24 mL) at 20-23° C. Add water (5 mL) to thesolution, followed by hemihydrate seed crystals (20 mg). Stir themixture for 2 h at 20-23° C., then cool to 0-5° C. Filter the mixture,wash with a solution of methanol (8 mL) and water (2 mL), and dry at50-60° C. under vacuum for 16 h to give 1.66 g of the title compound.

Example P-3 Biphenyl-4-carboxylic acid(R)-(6-(1-((4-fluorobenzyl)methylamino)ethylideneamino)-2(R)-hydroxyindan-1-yl)amidehemihydrate

Combine a solution of6-(1-((4-Fluorobenzyl)methylamino)ethylideneamino)-2-hydroxy-1-biphenylaminoindaneacetonitrile solvate (101 g) and methanol (1.2 L) with Darco G-60 (5 g).After stirring for 15-30 min at 15-25° C., filter the mixture and rinsethe filtered solids with methanol (0.4 L). Add water (0.4 L) to thecombined filtrate, rinse, and add hemihydrate seed crystals (1.5 g).Stir the mixture 2-3 h at 15-25° C., then cool to 0-5° C. and stiranother 90 min. Filter the mixture, wash with a 0-5° C. solution ofmethanol (0.8 L) and water (0.2 L), and dry at 47-53° C. under vacuumfor 20 h to give 88.7 g of the title compound.

The compounds of the present invention can be administered alone or inthe form of a pharmaceutical composition, that is, combined withpharmaceutically acceptable carriers or excipients, the proportion andnature of which are determined by the solubility and chemical propertiesof the compound selected, the chosen route of administration, andstandard pharmaceutical practice. The compounds of the presentinvention, while effective themselves, may be Formulated andadministered in the form of their pharmaceutically acceptable salts, forpurposes of stability, convenience, solubility, and the like. Inpractice, the compounds of Formula I are usually administered in theform of pharmaceutical compositions, that is, in admixture withpharmaceutically acceptable carriers or diluents.

Thus, the present invention provides pharmaceutical compositionscomprising a compound of Formula I and a pharmaceutically acceptablediluent. The present invention also provides suitable packaging,including a label, containing the pharmaceutical compositions comprisinga compound of Formula I.

The compounds of Formula I can be administered by a variety of routes.In effecting treatment of a patient afflicted with disorders describedherein, a compound of Formula I can be administered in any form or modewhich makes the compound bioavailable in an effective amount, includingoral and parenteral routes. For example, compounds of Formula I can beadministered orally, by inhalation, subcutaneously, intramuscularly,intravenously, transdermally, intranasally, rectally, occularly,topically, sublingually, buccally, and the like. Oral administration isgenerally preferred for treatment of the disorders described herein.

One skilled in the art of preparing Formulations can readily select theproper form and mode of administration depending upon the particularcharacteristics of the compound selected, the disorder or condition tobe treated, the stage of the disorder or condition, and other relevantcircumstances. (Remington's Pharmaceutical Sciences, 18th Edition, MackPublishing Co. (1990)).

The pharmaceutical compositions are prepared in a manner well known inthe pharmaceutical art. The carrier or excipient may be a solid,semi-solid, or liquid material which can serve as a vehicle or mediumfor the active ingredient. Suitable carriers or excipients are wellknown in the art. The pharmaceutical composition may be adapted fororal, inhalation, parenteral, or topical use and may be administered tothe patient in the form of tablets, capsules, aerosols, inhalants,suppositories, solutions, suspensions, or the like.

The compounds of the present invention may be administered orally, forexample, with an inert diluent or capsules or compressed into tablets.For the purpose of oral therapeutic administration, the compounds may beincorporated with excipients and used in the form of tablets, troches,capsules, elixirs, suspensions, syrups, wafers, chewing gums and thelike. These preparations should contain at least 4% of the compound ofthe present invention, the active ingredient, but may be varieddepending upon the particular form and may conveniently be between 4% toabout 70% of the weight of the unit. The amount of the compound presentin compositions is such that a suitable dosage will be obtained.Preferred compositions and preparations according to the presentinvention may be determined by a person skilled in the art.

The tablets, pills, capsules, troches, and the like may also contain oneor more of the following adjuvants: binders such as microcrystallinecellulose, gum tragacanth or gelatin; excipients such as starch orlactose, disintegrating agents such as alginic acid, Primogel, cornstarch and the like; lubricants such as magnesium stearate or Sterotex;glidants such as colloidal silicon dioxide; and sweetening agents suchas sucrose or saccharin may be added or a flavoring agent such aspeppermint, methyl salicylate or orange flavoring. When the dosage unitform is a capsule, it may contain, in addition to materials of the abovetype, a liquid carrier such as polyethylene glycol or a fatty oil. Otherdosage unit forms may contain other various materials which modify thephysical form of the dosage unit, for example, as coatings. Thus,tablets or pills may be coated with sugar, shellac, or other coatingagents. A syrup may contain, in addition to the present compounds,sucrose as a sweetening agent and certain preservatives, dyes andcolorings and flavors. Materials used in preparing these variouscompositions should be pharmaceutically pure and non-toxic in theamounts used.

For the purpose of oral and parenteral therapeutic administration, thecompounds of the present invention may be incorporated into a solutionor suspension. These preparations typically contain at least 0.1% of acompound of the invention, but may be varied to be between 0.1 and about90% of the weight thereof. The amount of the compound of Formula Ipresent in such compositions is such that a suitable dosage will beobtained. The solutions or suspensions may also include one or more ofthe following adjuvants: sterile diluents such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl paraben; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylene diaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. Theparenteral preparation can be enclosed in ampoules, disposable syringesor multiple dose vials made of glass or plastic. Preferred compositionsand preparations are able to be determined by one skilled in the art.

The compounds of the present invention may also be administeredtopically, and when done so the carrier may suitably comprise asolution, ointment, or gel base. The base, for example, may comprise oneor more of the following: petrolatum, lanolin, polyethylene glycols,bees wax, mineral oil, diluents such as water and alcohol, andemulsifiers, and stabilizers. Topical Formulations may contain aconcentration of the Formula I or its pharmaceutical salt from about 0.1to about 10% w/v (weight per unit volume).

The compounds of Formula I are agonists of the M-1 muscarinic receptors.Moreover the compounds of Formula I are selective agonists of thatparticular muscarinic receptor. The compounds of the present inventionpossess particularly useful properties related to their bioavailability,pharmacokinetics, safety, and efficacy. Muscarinic agonists, includingtheir subtype binding profile, can be identified by the methods that arewell known in the art.

In one embodiment, the present invention provides methods of treatingdisorders associated with muscarinic receptors, comprising:administering to a patient in need thereof an effective amount of acompound of Formula I. Thus, the present invention contemplates thevarious disorders described to be treated herein and others which can betreated by such agonists as are appreciated by those skilled in the art.

A number of the disorders which can be treated by muscarinic agonistsare known according to established and accepted classifications, whileothers are not. For example, cognition is a complicated and sometimespoorly defined phenomenon. It is, however, widely recognized thatcognition includes various “domains.” These domains include short termmemory, long term memory, working memory, executive function, andattention.

It is understood that the compounds of the present invention are usefulfor treatment of disorders characterized by a deficit in any of thecognitive domains listed above or in other aspects of cognition. Thusthe term “cognitive disorders” is meant to encompass any disordercharacterized by a deficit in one or more cognitive domain, includingbut not limited to short term memory, long term memory, working memory,executive function, and attention.

One cognitive disorder to be treated by the present invention isage-related cognitive decline. This disorder is not well defined in theart, but includes decline in the cognitive domains, particularly thememory and attention domains, which accompany aging. Another cognitivedisorder is mild cognitive impairment. Again, this disorder is not welldefined in the art, but involves decline in the cognitive domains, andis believed to represent a group of patients the majority of which haveincipient Alzheimer's disease. Another cognitive disorder is cognitiveimpairment associated with schizophrenia. The relationship betweencognitive disturbances and other symptoms of schizophrenia is notclearly understood at present. It has been observed that some peopleexperience cognitive problems much before they develop positivesymptoms, while others acquire cognitive deterioration after the firstepisode and with subsequent relapses. Yet another cognitive disorder ischemotherapy-induced cognitive impairment. People who undergo cancerchemotherapy may experience a decline in cognitive function and thisdecline can be long lasting. Also, a wide variety of insults, includingstroke, ischemia, hypoxia, inflammation, infectious processes andcognitive deficits subsequent to cardiac bypass surgery and grafting,stroke, cerebral ischemia, spinal cord trauma, head trauma, perinatalhypoxia, fetal alcohol syndrome, cardiac arrest, and hypoglycemicneuronal damage, vascular dementia, multi-infarct dementia, amylotrophiclateral sclerosis, chemotherapy, and multiple sclerosis can result incognitive deficits as a sequella which can be treated according to thepresent invention.

Where the disorders which can be treated by muscarinic agonists areknown according to established and accepted classifications, theseclassifications can be found in various sources. For example, atpresent, the fourth edition of the Diagnostic and Statistical Manual ofMental Disorders (DSM-IV™) (1994, American Psychiatric Association,Washington, D.C.), provides a diagnostic tool for identifying many ofthe disorders described herein. Also, the International Classificationof Diseases, Tenth Revision (ICD-10), provides classifications for manyof the disorders described herein. The skilled artisan will recognizethat there are alternative nomenclatures, nosologies, and classificationsystems for disorders described herein, including those as described inthe DSM-IV and ICD-10, and that terminology and classification systemsevolve with medical scientific progress.

In particularly preferred embodiments, the present invention providesmethods of treating disorders selected from the group consisting of:cognitive disorders (including age-related cognitive disorder, mildcognitive impairment, cognitive impairment associated withschizophrenia, and chemotherapy-induced cognitive impairment), ADHD,mood disorders (including depression, mania, bipolar disorders),psychosis (in particular schizophrenia and schizophreniform disorder),dementia (including Alzheimer's disease, AIDS-induced dementia, vasculardementia, and dementia lacking distinctive histology), Parkinson'sdisease, Huntington's Chorea, pain (including acute pain and chronicpain), xerostomia (dry mouth), Lewy body disease (including diffuse Lewybody disease), aphasia (including primary aphasia and primary aphasiasyndromes), aphasia (including primary aphasia and primary aphasiasyndromes), hypotensive syndromes, and chronic colitis (includingCrohn's disease), comprising: administering to a patient in need thereofan effective amount of a compound of Formula I. That is, the presentinvention provides for the use of a compound of Formula I orpharmaceutical composition thereof for the treatment disordersassociated with muscarinic receptors.

It is recognized that the terms “treatment” and “treating” are intendedto include improvement of the symptomatology associated with each of thedisorders associated with muscarinic receptors described herein. Also,it is also recognized that one skilled in the art may affect thedisorders by treating a patient presently afflicted with the disordersor by prophylactic ally treating a patient believed to be susceptible tosuch disorders with an effective amount of the compound of Formula I.Thus, the terms “treatment” and “treating” are intended to refer to allprocesses wherein there may be a slowing, interrupting, arresting,controlling, or stopping of the progression of the disorders describedherein, but does not necessarily indicate a total elimination of allsymptoms, and is intended to include prophylactic treatment of suchdisorders.

It is understood that the present invention includes adjunctivetreatment of the disorders described herein. More specifically, thecompounds of Formula I are useful to treat disorders in which acognitive deficit is one of the symptoms in combination with a widevariety of other therapeutic agents, in particular, in combination withAMPA potentiators; with typical and atypical antipsychotics, includingolanzapine; with a variety of agents such as mGluR agonists, with NMDAantagonists, with IL 1-6 inhibitors, with other cholinergics, includingcholinesterase inhibitors, such as tacrine and donepezil, and compoundsthat inhibit amyloid protein processing, including inhibitors of amyloidprecursor protein processing and antibodies directed against amyloidproteins; with antidepressants, including SSRIs and SNRIs such asfluoxetine, paroxetine, and venlafaxine; and with anxiolytic agents;etc. It is believed that the combinations above are synergisticallybeneficial providing efficacy at doses that are a small fraction ofthose required to produce the same effect with the individualcomponents.

In accordance with the adjunctive treatments, described above, thepresent invention also provides a product containing a compound ofFormula I and one or more therapeutic agents selected from the groupconsisting of AMPA potentiators; typical and atypical antipsychotics,including olanzapine; mGluR agonists; NMDA antagonists; IL 1-6inhibitors; cholinesterase inhibitors, such as tacrine and donepezil;compounds that inhibit amyloid protein processing, including inhibitorsof amyloid precursor protein processing and antibodies directed againstamyloid proteins; antidepressants, including SSRIs and SNRIs such asfluoxetine, paroxetine, and venlafaxine; and anxiolytic agents as acombined preparation for simultaneous, separate or sequentialadministration in the treatment of disorders in which a cognitivedeficit is one of the symptoms. In another embodiment the presentinvention also provides for the use of a compound of Formula I togetherwith one or more therapeutic agents selected from AMPA potentiators;typical and atypical antipsychotics, including olanzapine; mGluRagonists; NMDA antagonists; IL 1-6 inhibitors; cholinesteraseinhibitors, such as tacrine and donepezil; compounds that inhibitamyloid protein processing, including inhibitors of amyloid precursorprotein processing and antibodies directed against amyloid proteins;antidepressants, including SSRIs and SNRIs such as fluoxetine,paroxetine, and venlafaxine; and anxiolytic agents for the manufactureof a medicament as a combined preparation for simultaneous, separate orsequential administration in the treatment of disorders in which acognitive deficit is one of the symptoms.

As used herein, the term “simultaneous, separate or sequentialadministration” means that the two or more therapeutic agents areadministered within a time frame which ensures that all of thetherapeutic agents will provide some therapeutic activity at aparticular point in time. That is to say, the therapeutic activitiesshould at least overlap to some degree although they need not becoterminus.

As used herein, the term “patient” includes a mammal which is afflictedwith one or more disorders associated with muscarinic receptors. It isunderstood that guinea pigs, dogs, cats, rats, mice, horses, cattle,sheep, pigs, and humans are examples of animals within the scope of themeaning of the term.

As used herein, the term “effective amount” of a compound of Formula Irefers to an amount, that is, the dosage which is effective in treatingthe disorders described herein.

An effective amount can be readily determined by the attendingdiagnostician, as one skilled in the art, by the use of conventionaltechniques and by observing results obtained under analogouscircumstances. In determining an effective amount, the dose of acompound of Formula I, a number of factors are considered by theattending diagnostician, including, but not limited to: the compound ofFormula I to be administered; the co-administration of other therapies,if used; the species of mammal; its size, age, and general health; thespecific disorder involved; the degree of involvement or the severity ofthe disorder; the response of the individual patient; the mode ofadministration; the bioavailability characteristics of the preparationadministered; the dose regimen selected; the use of other concomitantmedication; and other relevant circumstances.

An effective amount of a compound of Formula I is expected to vary fromabout 0.01 milligram per kilogram of body weight per day (mg/kg/day) toabout 50 mg/kg/day, and preferable from 0.1 milligram per kilogram ofbody weight per day (mg/kg/day) to about 20 mg/kg/day. More preferredamounts can be determined by one skilled in the art.

Of the disorders to be treated according to the present invention anumber are particularly preferred. Particularly preferred disordersinclude the treatment of cognitive disorders (particularly mildcognitive impairment and cognitive impairment associated withschizophrenia), Alzheimer's disease, and psychosis, includingschizophrenia.

A number of preclinical laboratory animal models have been described forthe disorders described herein.

EXAMPLE A

Radial Arm Maze

The delayed non-match to sample task has been used to study the effectof drugs on memory retention (Pussinen, R. and Sirvio, J. J ofPsychopharm 13: 171-179(1999); Staubli, U., et al. Proc Natl Acad Sci91: 777-781(1994)) in the eight arm radial maze.

Well-trained rats were allowed to retrieve food rewards from fourrandomly selected arms of the maze (sampling phase). Some time later,the rats were exposed to eight open arms and were tested for theirability to remember and avoid the arms they had previously entered toobtain food. Re-entry into an arm that was baited during the samplingsession was counted as a reference error, whereas entry into the samearm more than once during the retention session was counted as workingerror. The total (reference +working) number of errors made during theretention test increases with increasing delay periods. For example,young male rats made 0.66 (+0.4) errors at a 1 minute delay, 2 (+0.5)errors at a one hour delay, and 3.95 (+0.2) errors at a seven hour delay(observations of this lab).

Male Sprague-Dawley rats were individually housed and maintained on a 12h light-dark cycle (lights on at 6 am). The rats were given free accessto water and maintained at 85% of their free-feeding weight bysupplemental feedings of Purina Lab Chow.

The rats were initially trained to search for food at the end of each ofthe eight arms. Once the rats had reached the criteria of no more thantwo errors (i.e. entering the same arm more than once during a session)on three consecutive days, a delay of one minute was imposed between thefourth and the fifth arm choices. This training ensured that the ratswere thoroughly familiar with the procedural aspects of the task beforeany drugs were administered. Once stable performance had been obtainedon the delay task (i.e. no more than one error was made on threeconsecutive days), drug and vehicle tests commenced using a seven hourdelay period. A novel set of arms was baited each day for each rat andthe maze was thoroughly cleaned during the delay period.

During the sampling session, each rat was placed on the center platformwith access to all eight arms of the maze blocked. Four of the eightarms were randomly selected and baited with food. The gates of thebaited arms were raised and the rat was allowed five minutes to obtainthe food at the end of each of the four arms. As soon as the rat hadobtained the food, it was removed, administered vehicle or various dosesof compounds, and placed back in its home cage. Seven hours later(retention session), the rat was placed back onto the center platformwith access to all eight arms blocked. The four arms that werepreviously baited during the sampling session, were baited and the gatesto all eight arms were raised. The rat was allowed five minutes toobtain the remaining four pieces of food. An entry into a non-baited armor a re-entry into a previously visited arm was counted as an error.Significance (p<0.05) was determined using a repeated measure ANOVAfollowed by a Dunnett's test for comparison with control.

In order to compare test compounds with standards, scopolamine andtacrine were administered s.c. immediately after the sampling phase. Theeffects of scopolamine, a known amnesic, were tested after a three-hourdelay, whereas the effect of tacrine, a cholinesterase inhibitor used inthe treatment of Alzheimer's disease was tested after a six-hour delay.Scopolamine disrupted retention after a three-hour delay in adose-related fashion. Tacrine significantly improved retention after asix-hour delay at 10, but not at 3 mg/kg.

EXAMPLE B

Acquisition in the Radial Maze 8-Arm Radial Maze Acquisition

A prominent early feature of Alzheimer's disease (AD) symptomology is apronounced deficit in declarative memory (R. W. Parks, R. F. Zec & R. S.Wilson (Eds.), Neuropsychology of Alzheimer's disease and otherdementias. NY: Oxford University Press pp. 3-80 (1993).

As the disease progresses, other domains of cognition become severelyaffected as well. Among the brain regions affected early in theprogression of Alzheimer's disease is the hippocampus, which is acritical neural substrate for declarative memory. Differences in thepattern of hippocampal neuronal loss in normal aging and Alzheimer'sdisease. Lancet, 344: 769-772(1994). One behavioral test that is oftenused to assess hippocampal function in animal models is the 8-arm radialmaze (Olton D. S. The radial arm maze as a tool in behavioralpharmacology. Physiology & Behavior, 40: 793-797 (1986)).

Lesions or pharmacological blockade of the hippocampus disruptperformance of this task. Moreover, aged animals generally show deficitsin this task (Porsolt R. D., Roux S. & Wettstein J. G. Animal models ofdementia. Drug Development Research, 35: 214-229(1995)).

In this test of spatial learning and memory, a hungry rat is placed inthe center of the maze and allowed to traverse the maze in search offood located at the end of each runway arm. In this version of the maze,the rat learns a win-shift strategy in which a visited arm is notreplaced. Therefore, the most efficient foraging strategy is to visiteach arm once. The version of the maze also taps into general learningprocesses as the rat is naïve to the maze on day one of the four dayexperiment.

Upon arrival, male Sprague Dawley®, rats were individually housed in aregular light-cycle colony room and allowed to acclimate for at least 4days prior to testing. Each rat was reduced to and maintained at 85% oftheir target body weight throughout the experiment. Proper body weightwas maintained by adjusting the allotment of lab chow based on acombination of age and the rat's daily bodyweight reading.

A session began with an individual rat being placed into the hub of themaze and then all guillotine doors were raised, allowing free access toall areas of the maze. A food hopper was located at the end of each ofthe 8 runway arms and a single food pellet was placed in each foodhopper. Each daily session terminated when either all 8 food-hoppers hadbeen visited or when the rat timed out (15 min on Day 1: 5 min on Days2-4). The number of arm entries was recorded. Errors were counted asrepeat arm entries or failures to visit an arm in the session period. Ananimal was excluded from the study if it failed to visit at least onearm on Day 1, 2 arms on Day 2, and at least 4 arms on Days 3 & 4.

Each rat was pseudo-randomly assigned to either a vehicle or drug groupand received the same treatment throughout the experimental period.Vehicle consisted of 5% acacia within sterile water. Injections wereadministered subcutaneously 20-30 minutes prior to each daily session.

In this acquisition task, vehicle-treated animals do not consistentlyshow significant acquisition of maze learning as compared to the numberof errors committed on Day 1. We have found that in compounds thatfacilitate acquisition of maze learning, the effects are often notobserved until the fourth day of training. Therefore, results consistedof total Day 4 errors across treatment groups.

EXAMPLE C

Functional Mobilization of Intracellular Calcium

CHO cells expressing muscarinic subtypes (M1-M5) are grown as monolayersin DMEM:F-12 (3:1), 10% FBSnz, 20 mM HEPES, 1% pen/strep, 250 μg/mL G418(GibcoBRL #10131-027). Cells are maintained under 95%/5% O₂/CO₂ andpassaged every 3-4 days. Cells are plated 24 hours in advance of theassay at a density of 50,000/well and 48 hours in advance at a densityof 25,000/well (100 μL/well) in Costar black-walled, clear-bottomed 96well plates (Costar #3603). Cells are then incubated with minimumessential medium containing the cytoplasmic Ca²⁺ indicator, Fluo-3 (1 mMFluo mixed 1:1 with 20% pluronic acid, then diluted to 5 μM finalconcentration in growth and supplemented with 2.5 mM, 50 μL/well) at 37°C. in an environment containing 5% CO₂ for 60 minutes. Cells are washedtwice with 100 μL/well of wash buffer containing Hanks Balanced SaltSolution (HBSS) without phenol red (1×) (GibcoBRL #14065-056), 20 mMHEPES (Sigma #P8761), and Probenecid (2.5 mM) (100×: 1:100). For theassay, 100 μL is added to each well (100 μL of 2×drug will be added bythe FLIPR). Plates are washed three times using a LabSystems multidropand residual buffer is removed. Plates are also blotted on paper towelsto remove remaining compound.

Compounds are prepared 2×(100 μL of drug added to 100 μL of assay bufferpresent in the well) in assay buffer containing 2% DMSO, HBSS withoutphenol red (1×) (GibcoBRL #14065-056), 20 mM HEPES (Sigma #P8761), andProbenecid (2.5 mM) (100×: 1:100).

The plates were then placed into a FLIPR instrument (fluorometricimaging plate reader system, Molecular Devices, Sunnyvale, Calif.) tomonitor cell fluorescence (λ_(EX)=488 nm, λ_(EM)=540 nm) before andafter the addition of compounds.

The selectivity of the M1 agonists arc evaluated by screening acrossother muscarinic receptor subtypes (M3 and M5) in a similar manner inboth the agonist and antagonist mode. Compounds are also sreened acrossa number of protein targets as well as the structurally related Gprotein-coupled receptor (GPCR) targets to insure selectivity for the M1receptor.

EXAMPLE D

Functional GTP Binding

Cell Culture: CHO cells transfected with human M1-M5 receptors weregrown either in suspension or in monolayer. For suspension culturescells were grown in roller bottles with constant agitation at 37° C. and5% CO₂ using Dulbecco's modified Eagles medium/F-12 (3:1) culture mediumsupplemented with 5% fetal bovine serum, 50 μg/ml tobramycin, and 20 mMHEPES. Monolayer cultures were grown in T-225 flasks at 37° C. and 5%CO₂ in Dulbecco's modified Eagles medium supplemented with 10% fetalbovine serum and 100,000 U/liter of penicillin/streptomycin. Cells wereharvested using trypsin-free dissociation media at 95% confluence andwere collected by centrifugation and stored at 80° C. Cells stablyexpressing human muscarinic receptors were obtained from the NationalInstitutes of Health.

Membrane Preparation: Cell pellets were thawed and resuspended in 20volumes of 20 mM sodium phosphate buffer, pH 7.4, and were homogenizedtwice for 30 seconds at high speed using a Tissuemizer. Homogenates werecentrifuged at 200 g for 15 min at 4° C. The supernatant was removed andreserved on ice. This procedure was repeated twice and the pooledsupernatants were then centrifuged at 40,000 g for 45 min at 4° C.Membranes were suspended at 5 mg protein/ml and were stored at 80° C.Unless indicated otherwise in the figure legends, membranes from M1, M2,and M4 cells were prepared from cells grown in suspension, whereas thosefrom M3 and M5 cells were from cells grown in monolayer. Receptordensities (pmol mg1 membrane protein) were 9.3, 0.7, 0.6, 0.9, and 4.8for M1-M5 receptors, respectively.

Striatal tissue from male Sprague-Dawley rats was homogenized by hand in10 volumes of 10 mM HEPES and 1 mM EGTA, pH 7.4, containing Completeprotease inhibitor cocktail, 1 mM dithiothreitol, and 10% sucrose. Thehomogenate was diluted 6-fold and centrifuged at 1000 g for 10 min at 4°C. The supernatant was saved and the pellet rehomogenized andcentrifuged as above. The combined supernatants were centrifuged at11,000 g for 20 min. The resulting pellet was homogenized in 40 volumesof 10 mM HEPES and 1 mM EGTA, pH 7.4, containing 1 mM dithiothreitol and1 mM MgCl₂, and was centrifuged at 27,000 g for 20 min. The resultingpellet was suspended in the same buffer at a protein concentration of1.5 mg/ml and aliquots were frozen and stored at 80° C.

GTPγ³⁵S Binding: Assays were run in 20 mM HEPES, 100 mM NaCl, and 5 mMMgCl₂ at pH 7.4 in a final volume of 200 μl in 96-well Costar plates at25° C. One hundred microliters of membrane preparation (25 μg proteinper well for cell membranes and 9-15 μg per well for brain membranes)containing the appropriate concentration of GDP was added followed byaddition of 50 μl of buffer ±agonists and antagonists being testedfollowed by 50 μl of GTPγ³⁵S to provide a final concentration in theassay of 200 pM for CHO membranes and 500 pM for brain membranes. ForCHO membranes, 0.1 μM GDP was used for M1, M3, and M5 receptor assays,whereas 1 μM GDP was used for M2 and M4 assays. For brain membranes 0.1μM GDP was used in assays carried out with anti-Gαq/11, whereas 50 μMGDP was used for assays using anti-Gαi(1-3) and anti-Gαo. CHO cellmembranes were incubated for 30 min at 25° C. with agonists andantagonists followed by addition of GTPγ³⁵S and incubation for anadditional 30 min. Brain membranes were incubated for 20 min at 25° C.with agonists and antagonists followed by addition of GTPγ³⁵S andincubation for an additional 60 min. Preincubation was employed toensure that agonists and antagonists were at equilibrium during thelabeling period.

To determine total membrane binding, 50 μl of suspended wheat germagglutinin (WGA)-coated SPA beads was added. After 15 min, plates werecentrifuged at 1000 g for 15 min and radioactivity was determined usinga Wallac plate counter. For determining binding to specific G proteins,³⁵S-labeled membranes were solubilized for 30 min with 0.27% NonidetP-40 (20 μl/well of a solution containing 1.5 ml of 10% Nonidet P-40 forevery 3.5 ml assay buffer) followed by addition of desired antibody (10μl/well) to provide a final dilution of 1/400 to 1/100 and incubationfor an additional 60 min. Fify microliters of suspended anti-IgG-coatedSPA beads was added per well, plates were incubated for 3 h, and thenwere centrifuged and radioactivity determined as above. Each bottle ofWGA-coated SPA beads was suspended in 10 ml of assay buffer and eachbottle of anti-IgG-coated SPA beads was suspended in 20 ml of assaybuffer. Protein was determined using the bicinchoninic acid assay.

Materials: ³⁵S-GTPγS (1000-1200 Ci/mmol), anti-rabbit-IgG andanti-mouse-IgG-coated SPA beads, and WGA-coated SPA beads were obtainedfrom Amersham (Arlington Heights, Ill.). Rabbit anti-Gαq/11 and rabbitanti-Gαi(1-3) were from Santa Cruz Biotechnologies (Santa Cruz, Calif.).Mouse monoclonal anti-Gαo was from Chemicon (Temecula, Calif.).Oxotremorine M and pirenzepine were from Research Biochemicals Inc.(Natick, Mass.).11-{[2-((Diethylamino)methyl)-1-piperidinyl]acetyl}-5,11-dihydro-6H-pyrido[2,3b][1,4]benzodiazepin-6-one (AFDX 116) was synthesized at Eli Lilly.Complete protease inhibitor cocktail and 10% Nonidet P-40 were fromBoehringer Mannheim (Indianapolis, Ind.).

The selectivity of the M1 agonists are evaluated by screening acrossother muscarinic receptor subtypes (M2 and M4) in both the agonist andantagonist mode. Compounds are also screened across a number of proteintargets as wail as the structurally related G protein-coupled receptor(GPCR) targets to insure selectivity for the M1 receptor.

1. A compound of the Formula

wherein Q, X, Y, and Z are independently selected from the groupconsisting of CR¹ and N, provided that no more than two of Q, X, Y, andZ are N and at least two of Q, X, Y, and Z are CH; or Y is CH, Z is CH,and the moiety “Q=X” represents “S” to form a thiophene ring; R¹ isindependently at each occurrence selected from the group consisting ofhydrogen, halogen, C₁-C₄ alkoxy, and C₁-C₄ alkyl; R² is selected fromthe group consisting of halogen; C₁-C₄ alkoxy; C₁-C₄ alkyl; C₃-C₈cycloalkyl; cyano; trifluoromethyl; pyridinyl optionally substitutedwith one to two substituents independently selected from the groupconsisting of halogen, C₁-C₄ alkoxy, and C₁-C₄ alkyl; thienyl optionallysubstituted with one substituent selected from the group consisting ofhalogen, C₁-C₄ alkoxy, and C₁-C₄ alkyl; phenyl optionally substitutedwith from one to three substituents independently selected from thegroup consisting of halogen, C₁-C₄ alkoxy, C₁-C₄ alkyl, trifluoromethyl,and cyano; and pyrrolyl optionally substituted with one to twosubstituents independently selected from the group consisting ofhalogen, C₁-C₄ alkoxy, and C₁-C₄ alkyl; R³ is selected from the groupconsisting of phenyl optionally substituted with one to threesubstituents independently selected from the group consisting ofhalogen, C₁-C₄ alkoxy, C₁-C₄ alkyl, trifluoromethyl, cyano, and nitro;naphthyl optionally substituted with one to three substituentsindependently selected from the group consisting of halogen, C₁-C₄alkoxy, C₁-C₄ alkyl, trifluoromethyl, cyano, and nitro; heteroaryloptionally substituted with one or two substituents independentlyselected from the group consisting of halogen, C₁-C₄ alkoxy, and C₁-C₄alkyl; or 1,3-benzodioxolyl optionally substituted with one substituentselected from the group consisting of halogen, C₁-C₄ alkoxy, and C₁-C₄alkyl; R⁴ is selected from the group consisting of hydrogen, hydroxy,and fluoro; R⁵ is selected from the group consisting of hydrogen,halogen, C₁-C₄ alkoxy, and C₁-C₄ alkyl; R^(a) is selected from the groupconsisting of hydrogen and methyl; t is one, two, or three; and m isone; or pharmaceutically acceptable addition salts thereof.
 2. Acompound according to claim 1 wherein R^(a) is methyl, R⁵ is hydrogen,R⁴ is hydroxy, t is one, m is one, and which has the transstereochemistry at the 1- and 2-position shown below:


3. A compound according to claim 1 wherein Q, X, Y, and Z are each CH.4. A compound according to claim 1 wherein R² is phenyl.
 5. A compoundaccording to claim 2 wherein R³ is phenyl substituted once with halogen.6. The compound biphenyl-4-carboxylic acid (R)-(6-(1-((4-fluorobenzyl)methylamino)ethylideneamino)-2(R)-hydroxyindan-1-yl)amideand the pharmaceutically acceptable salts thereof.
 7. A pharmaceuticalcomposition comprising a compound of claim 1 and one or morepharmaceutically acceptable carriers, excipients, or diluents.
 8. Amethod of treating Alzheimer's disease, comprising: administering to apatient in need thereof an effective amount of a compound of claim
 1. 9.A method of treating schizophrenia, comprising: administering to apatient in need thereof an effective amount of a compound of claim 1.10. A pharmaceutical composition comprising a compound of claim 2 andone or more pharmaceutically acceptable carriers, excipients, ordiluents.
 11. A method of treating Alzheimer's disease, comprising:administering to a patient in need thereof an effective amount of acompound of claim
 2. 12. A method of treating schizophrenia, comprising:administering to a patient in need thereof an effective amount of acompound of claim
 2. 13. A method of treating Lewy body diseasecomprising: administering to a patient in need thereof an effectiveamount of a compound of claim
 1. 14. A method of treating Lewy bodydisease comprising: administering to a patient in need thereof aneffective amount of a compound of claim 2.